Differential Phosphorylation of Perilipin 1A at the
Initiation of Lipolysis Revealed by Novel Monoclonal
Antibodies and High Content Analysis
Patrick M. McDonough1*, Dominique Maciejewski-Lenoir1., Sean M. Hartig2., Rita A. Hanna1,
Ross Whittaker1, Andrew Heisel1, James B. Nicoll3, Benjamin M. Buehrer3, Kurt Christensen2,
Maureen G. Mancini2, Michael A. Mancini2, Dean P. Edwards2, Jeffrey H. Price1,4
1Vala Sciences Inc, San Diego, California, United States of America, 2Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, United
States of America, 3Zen-Bio, Inc, Research Triangle Park, North Carolina, United States of America, 4Signal Transduction Program, The Sanford-Burnham Institute for
Medical Research, La Jolla, California, United States of America
Lipolysis in adipocytes is regulated by phosphorylation of lipid droplet-associated proteins, including perilipin 1A and
hormone-sensitive lipase (HSL). Perilipin 1A is potentially phosphorylated by cAMP(adenosine 39,59-cyclic monophosphate)-
dependent protein kinase (PKA) on several sites, including conserved C-terminal residues, serine 497 (PKA-site 5) and serine
522 (PKA-site 6). To characterize perilipin 1A phosphorylation, novel monoclonal antibodies were developed, which
selectively recognize perilipin 1A phosphorylation at PKA-site 5 and PKA-site 6. Utilizing these novel antibodies, as well as
antibodies selectively recognizing HSL phosphorylation at serine 563 or serine 660, we used high content analysis to
examine the phosphorylation of perilipin 1A and HSL in adipocytes exposed to lipolytic agents. We found that perilipin PKA-
site 5 and HSL-serine 660 were phosphorylated to a similar extent in response to forskolin (FSK) and L-c-melanocyte
stimulating hormone (L-c-MSH). In contrast, perilipin PKA-site 6 and HSL-serine 563 were phosphorylated more slowly and
L-c-MSH was a stronger agonist for these sites compared to FSK. When a panel of lipolytic agents was tested, including
multiple concentrations of isoproterenol, FSK, and L-c-MSH, the pattern of results was virtually identical for perilipin PKA-site
5 and HSL-serine 660, whereas a distinct pattern was observed for perilipin PKA-site 6 and HSL-serine 563. Notably, perilipin
PKA-site 5 and HSL-serine 660 feature two arginine residues upstream from the phospho-acceptor site, which confers high
affinity for PKA, whereas perilipin PKA-site 6 and HSL-serine 563 feature only a single arginine. Thus, we suggest perilipin 1A
and HSL are differentially phosphorylated in a similar manner at the initiation of lipolysis and arginine residues near the
target serines may influence this process.
Citation: McDonough PM, Maciejewski-Lenoir D, Hartig SM, Hanna RA, Whittaker R, et al. (2013) Differential Phosphorylation of Perilipin 1A at the Initiation of
Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis. PLoS ONE 8(2): e55511. doi:10.1371/journal.pone.0055511
Editor: Sally Martin, The University of Queensland, Australia
Received February 28, 2012; Accepted January 2, 2013; Published February 6, 2013
Copyright: ? 2013 McDonough et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported, in part, by United States National Institutes of Health (NIH)/NIDDK (National Institute of Diabetes and Digestive and
Kidney Diseases) 5R44DK074333 (P. M. McDonough). S. M. Hartig was supported by NIH/NIDDK 1F32DK85979. Imaging resources were supported by the John S.
Dunn Gulf Coast Consortium for Chemical Genomics (M. A. Mancini), Specialized Cooperative Centers for Reproduction Grant NIH/NICHD (National Institute of
Child Health and Human Development) U54 HD-007495 (B. W. O’Malley), Texas Gulf Coast Digestive Diseases Center Grant NIH/NIDDK P30 DK-56338 (M. K. Estes),
Public Health Services Cancer Center Support Grant NIH/NCI P30 CA-125123 (C. K. Osborne), and the Dan L. Duncan Cancer Center of Baylor College of Medicine.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors P. M. McDonough, R. Whittaker, A. Heisel, and J. H. Price are employees of Vala Sciences Inc. P. M. McDonough and J. H. Price
are also stock-holders in Vala Sciences Inc. D. Maciejewski-Lenoir volunteered for Vala Sciences Inc for this project. J. B. Nicoll and B. M. Buehrer are employees of
Zen-Bio, Inc. Reagents and software referred to in this paper are available from Vala Sciences Inc as commercial products. This does not alter the authors’
adherence to all the PLOS ONE policies on sharing data and materials.
* E-mail: email@example.com
. These authors contributed equally to this work.
Lipid droplets are cellular organelles consisting of a neutral lipid
core of triacylglycerides (TAGs), surrounded by a phospholipid
membrane and a suite of proteins, which regulate lipid metabolism
. Lipolysis is a key metabolic process whereby TAGs in the lipid
droplets are processed by lipases to release fatty acids for ß-
oxidation. A current model for the initiation of lipolysis  in
adipocytes is presented in Figure 1. The protein perilipin 1A
(PLIN1) was the founding member of the five gene perilipin family
. Perilipin 1A is tightly associated with the cytoplasmic side of
the lipid droplets in adipocytes [4,5,6]. Under basal conditions,
perilipin 1A may inhibit lipolysis by blocking lipase access to
TAGs and/or by sequestering CGI-58 (also known as Abhd5) ,
while HSL is largely located in the cytoplasm. Agents that increase
c-AMP activate c-AMP-dependent protein kinase (PKA) to
phosphorylate perilipin 1A and HSL. Perilipin 1A phosphoryla-
tion releases CGI-58, which enables CGI-58 to activate adipose
triglyceride lipase (ATGL). ATGL is the initiating lipase in
lipolysis, as it removes a fatty acid moiety from TAG to form
diacylglycerol. Additionally, phosphorylated HSL translocates
from the cytoplasm to the lipid droplets where it interacts with
phosphorylated perilipin 1A [8,9]. HSL acts as the second lipase in
PLOS ONE | www.plosone.org1 February 2013 | Volume 8 | Issue 2 | e55511
the pathway, where it releases a fatty acid moiety from
diacylglycerol to form monoacylglycerol. Monoacylglycerol is also
further processed by monoacylglycerol lipase to form fatty acid
and glycerol (not shown).
While it is clear that lipolysis is orchestrated by phosphorylation
of several proteins, the specific relationship between phosphory-
lation events is not well understood. Murine perilipin 1A (National
Center for Biotechnology Information (NCBI) reference sequence
NP_783571.2) has six potential PKA phosphorylation sites 
located at serines 81, 222, 276, 433, 492, and 517, referred to as
PKA-sites 1–6, respectively ; splice variants of perilipin 1
(PLIN1b and PLIN1c), which lack PKA sites 4–6, are also less
commonly expressed . Human perilipin 1A (NCBI reference
sequence NP_001138783.1) is similar to murine perilipin 1A, but
lacks PKA-site 2. Furthermore, there are minor insertions and
deletions between the amino acid sequences for perilipin 1A
between the two species, making serines 497 and 522 of human
perilipin 1A equivalent to serines 492 and 517 of murine perilipin
1A. PKA-sites 5 and 6 are likely to be critical for proper regulation
of lipolysis. PKA-site 5 promotes agonist-induced lipid droplet
dispersion , while PKA-site 6 maximizes activation of ATGL-
dependent lipolysis . The timing and extent of phosphoryla-
tion of perilipin 1A at these sites is unknown.
HSL is phosphorylated on serines 563, 659, and 660 by PKA at
the initiation of lipolysis. Phosphorylation of HSL on serines 659
and 660 are activating, but the function of phosphorylation on
serine 563 is unclear . Additionally, HSL can be phosphor-
ylated on serine 600 by extracellular signal-regulated kinase/
mitogen-activated protein kinase (ERK-MAPK) which increases
its activity , whereas phosphorylation of HSL on serine 565 by
AMP-activated protein kinase, (AMPK) is inhibitory . Recent
work by Martin et al.  established the early time course and
spatial distribution of HSL phosphorylation in murine 3T3L1
adipocytes, utilizing antibodies specific to HSL phosphorylation at
either serine 563 (pHSL-serine 563) or serine 660 (pHSL-serine
660). Within a few seconds of exposure to FSK, pHSL-serine 660
appears near the plasma membrane, preceding translocation of
HSL to lipid droplets. In contrast, pHSL-serine 563 appears with a
delay relative to pHSL-serine 660 and is localized exclusively at
the edges of lipid droplets. Collectively, it was proposed that
separate pools of PKA, proximal to either the plasma membrane
or lipid droplets, may orchestrate the differential phosphorylation
of HSL at serine 660 and serine 563 .
The goal of the present study was to examine the specificity and
time course of perilipin 1A phosphorylation at the initiation of
lipolysis. Towards this end, monoclonal antibodies were developed
which selectively label perilipin 1A phosphorylation at either
PKA-site 5 (murine serine 492/human serine 497) or PKA-site 6
(murine serine 517/human serine 522). We utilized these reagents,
in combination with high content analysis, to quantify the
appearance of phospho-perilipin and phospho-HSL in adipocytes
following exposure to lipolytic agents. We used FSK and
isoproterenol, which increase cAMP, and L-c-MSH, a peptide
hormone that induces lipolysis by an incompletely characterized
mechanism [18,19], as model lipolytic agents. Our study reveals
perilipin 1A is phosphorylated on PKA-site 5 in a manner similar
to HSL-serine 660 (relatively rapid, and with equal efficacy by
FSK and L-c-MSH) whereas perilipin 1A PKA-site 6 is
phosphorylated in a manner more similar to HSL-serine 563
(more slowly, with L-c-MSH a stronger agonist than FSK).
Comparison of the sequences surrounding the phosphorylation
sites on perilipin and HSL suggests the presence of single or
multiple arginine residues upstream from the phospho-acceptor
serine may influence the differential phosphorylation of perilipin
1A and HSL at the initiation of lipolysis.
Materials and Methods
For certain experiments, human cells were utilized, which were
obtained and de-identified by Zen-Bio, Inc, following written
informed consent from the donors, utilizing a protocol approved
by the Essex Institutional Review Board, Inc of Lebanon, New
Jersey (‘‘Surgical Waste Remnants’’ ZB02, 6/30/06, Amendment
No. 1, 3/14/11; this protocol was most recently approved, June
13, 2011, and runs through May 28, 2013).
3T3L1 cells were obtained from ATCC and maintained in
Growth Medium (Dulbecco’s Modified Eagle’s Medium supple-
mented with 10% fetal bovine serum, penicillin, streptomycin,
amphotericin). Cells were passed a maximum of 17 times prior to
Figure 1. Regulation of lipolysis in adipocytes. A, A current model for the hormonal regulation of lipolysis initiation is shown. Proteins depicted
include perilipin 1A (Peri), Hormone Sensitive Lipase (HSL), Adipocyte Triglyceride Lipase (ATGL), CGI-58, and PKA. Lipid species depicted include
triacylglycerol (TAG), diacylglycerol (DAG), monoacylglycerol (MAG), and fatty acid (FA). Under basal conditions, perilipin and HSL are
unphosphorylated and HSL is found throughout the cytoplasm. Stimulation of lipolysis involves activation of PKA, phosphorylation of perilipin
1A and HSL, release of CGI-58 from perilipin, binding of CGI-58 to ATGL, and translocation of HSL to perilipin. TAG is sequentially processed to DAG by
ATGL and to MAG by HSL with FA released at each step. B, Amino acid sequences are shown for perilipin 1A PKA site 5, and PKA site 6, and for HSL
serine 563 and serine 660. The target serine in each sequence is underlined.
Differential Phosphorylation of Perilipin 1A
PLOS ONE | www.plosone.org2 February 2013 | Volume 8 | Issue 2 | e55511
use in experiments. To initiate adipogenesis, 3T3L1 cells were
seeded in 75 cm2flasks and allowed to grow to confluence. The
cells were then exposed to Differentiation Media (Growth Medium
additionally supplemented with 0.5 mM IBMX (3-Isobutyl-1-
methylxanthine), 0.25 mM dexamethasone, and 1 mg/ml insulin).
After 3 or 4 days in Differentiation Medium, the cells were
harvested, seeded in 96-well glass bottomed dishes (Nunc Cat.
No 73520-168, two 96-well dishes/flask) precoated with gelatin
which was cross-linked with glutaraldehyde  at a density of
67,000 cells/well, and re-fed with Growth Medium. Experiments
were performed 2 to 5 days after seeding into the 96-well dishes.
With this protocol, a high degree of the cells typically featured
clusters of lipid droplets. HeLa cells were also obtained from
Human subcutaneous preadipocytes (a mixture of preadipocytes
from 6 females, average age 39, average BMI=0.273) were
obtained via liposuction by Zen-Bio (Research Triangle Park, NC)
and were plated (13,000 cells/well, passage 2) on glass-bottomed
96-well dishes precoated with gelatin that was cross linked with
glutaraldehyde. To induce differentiation of the cells to adipocytes,
the cells were exposed to Differentiation Medium (DM-2, Zen-
Bio), which contains factors that induce adipogenesis in this cell
type; alternatively, in certain experiments, the cells were exposed
to a similar media, which featured the peroxisome proliferator-
(300 nM), which is also effective at inducing differentiation.
Plasmids and Reagents
The cDNA sequence encoding human perilipin 1A was
purchased from Origene (Rockville, MD). S497A and S522A
mutations were introduced by using internal primers including the
sequence encoding each serine. The codon for serine was altered
to encode alanine. Subsequently, the wild-type or mutated
perilipin 1A cDNAs were transferred to either GFP or mCherry
vectors using appropriate restriction sites. Wild-type and mutated
forms of perilipin 1A were sequenced to verify fidelity. Antibodies
used to visualize HSL or perilipin 1A were from Vala Sciences Inc
(San Diego, California). GP29, a guinea pig polyclonal antibody
raised against perilipin, was from Progen (Heidelberg, Germany).
(equivalent to Bodipy 493/503TMLife Technologies) was used to
visualize lipid droplets. Lipofectamine 2000, AlexaFluor 647-anti
mouse secondary antibody, Cell Mask Blue, HCS LipidTox
DeepRed, and SlowFade Gold mounting solution were from Life
Technologies (Carlsbad, California). HiLyte FluorTM647 goat-
anti-rabbit (far-red fluorescent secondary antibody) was from
AnaSpec (Fremont, California). Texas Red conjugated goat-anti-
mouse (red fluorescent secondary antibody) was from Jackson
Production of Mouse Monoclonal Antibodies
Mouse monoclonal antibodies were developed and character-
ized by the Monoclonal Antibody Core Facility at Baylor College
of Medicine. Mice were inoculated with peptides containing
amino acid sequences identical to those surrounding PKA-site 5 or
PKA-site 6 of the human perilipin 1A sequence (Figure 1). N-a-
Fmoc-O-benzyl-L-phosphoserine was substituted for serine at the
phospho-acceptor site, to create a stable analogue of phospho-
serine. Hybridomas were prepared from sera-positive mice by
previously described techniques [21,22,23] and verified for the
production of antibodies against the immunizing peptide by
ELISA. The antibodies developed in this study, designated anti-
pPeri-site 5 and anti-pPeri-site 6, are available for purchase from
Vala Sciences Inc (catalogue items MAb #4855 and MAb #4856,
Characterization of Antibody Specificity
After isolation of hybridomas secreting monoclonal antibodies,
specificity to codon mutation was tested using wild-type and
mutant perilipin constructs in GFP and mCherry vectors,
respectively. HeLa cells were plated in 6 well plates 24 h prior
to transfection. HeLa cells were transfected using Lipofectamine
2000 with a single plasmid (either GFP-PLIN1 (wild type), mCh-
PLIN 1 S497A, or mCh-PLIN1 S522A) used per transfection
reaction. The next day, the cells were detached and seeded on
22 mm poly-D-lysine coated coverslips placed in 24-well dishes.
Approximately 8 hr later, oleic acid complexed to fatty acid-free
BSA (a kind gift from Larry Chan, Baylor College of Medicine,
550 mM) was added to induce lipid droplet formation. After an
overnight incubation with oleic acid, the cells were treated with
forskolin (24 mM) for 7 min to increase PKA activity. Treatments
and transfections were performed in OPTI-MEM (Invitrogen).
Following treatment with forskolin or DMSO, cells were washed
and fixed in 4% paraformaldehyde (ultrapure, Electron Micros-
copy Sciences, Hatfield, PA) for 30 minutes at room temperature.
Coverslips were quenched with 100 mM ammonium chloride and
washed three times with PIPES/HEPES/EGTA/MgCl2 (PEM)
buffer, prepared at a final pH of 6.8. Cells were permeabilized
with 0.2% Triton X-100 in PEM for 10 minutes, washed three
times with PEM and blocked in 5% milk in PEM/0.01% saponin
blocking solution for 30 minutes at room temperature. Antibodies
were then diluted in the blocking solution and incubated overnight
at 4uC. Subsequently, coverslips were washed with PEM and
incubated with AlexaFluor 647-conjugated anti-mouse secondary
antibodies (Life Technologies) for 1 h at room temperature. Cells
were again washed 3 times and incubated with CellMask Blue
(1 mg/ml), and DAPI (10 mg/ml) in PEM for 45 minutes at room
temperature. Dyes were then aspirated, PBS/0.01% azide added,
and cells imaged immediately. Coverslips were mounted with
SlowFade Gold (Invitrogen). Images were acquired with a
Beckman coulter IC-100 Image Cytometer equipped with a
Nikon S Fluor 20X/0.75NA objective or a DeltaVision Core
Image Restoration Microscope (Applied Precision, Issaquah, WA)
using a 60X, 1.42 NA Plan Achromat objective (Olympus, Center
Valley, PA), and a Photometrics CoolSnap HQ2 CCD camera.
Preadipocytes were obtained from Zen Bio (SP-F-SL, lot#
SL0048) and were plated directly on 96 well plate at the density of
15000 cells per well. siRNA transfection was performed at the time
of plating. A mix of the cells, media (DMEM, Gibco 11966-25
500 ml, lot# 1095492), siRNA (SilencerH Select, Life Technolo-
gies), and transfection reagent (RNAI max, Invitrogen, 13778-075,
lot# 804296) was prepared, and 200 ml of the mixture was added
per well. The sense sequence of the perilipin 1A siRNA was
GCACAUACCCUGCAGAAGA (59 to 39); additionally, an
siRNA that does not correspond to a human gene was utilized
as a control. The perilipin 1A and control siRNAs were tested at
four different concentrations: 0 nM, 10 nM, 20 nM, and 50 nM.
48 hours post transfection, transfection media was removed, and
differentiation medium was added for 6 days. Cells were then
treated with forskolin at a final concentration of 6 mM in DMEM
for 30 minutes, then fixed and labeled for nuclei, lipid droplets,
and primary antibodies (anti-pPeri-ser 5, anti-pPeri-ser6, and
GP29) via methods described below.
Differential Phosphorylation of Perilipin 1A
PLOS ONE | www.plosone.org3February 2013 | Volume 8 | Issue 2 | e55511
Cells were plated on 96-well dishes and exposed to experimental
treatments, after which they were fixed with 4% paraformalde-
hyde plus 0.025% glutaraldehyde. The cells were then permea-
bilized with 0.1% Triton-X-100 (20 minutes) and blocked for 60
minutes in blocking buffer (10% goat serum, 3% BSA, 0.02%
sodium azide in PBS). Fixed cells were labeled with primary
antibodies diluted in blocking buffer for either 60 minutes at 37 C
with rotation, or overnight at 4 C. The samples were then rinsed
3X with PBS, incubated for one hour with appropriate secondary
antibodies (ether goat- anti-rabbit, goat- anti-mouse, or goat-anti-
guinea pig) conjugated to red channel or far-red channel
fluorophores, along with the Lipid Droplet Stain to label lipid
droplets in the green channel. Following incubation with the
secondary reagents, the plates were rinsed 3X with PBS. DAPI
was then added; the dishes then sealed and incubated a further 20
minutes at room temperature, prior to automated image
acquisition. In experiments in which peptides were tested for their
ability to block primary antibody binding, the primary antibodies
were preincubated with 0 to 10 mg peptide in a volume of 300 ml
for 30 minutes at 37 C.
Conventional Fluorescence Microscopy
Images were acquired with a Beckman Coulter IC 100 using
established methods . This instrument includes a Nikon
Eclipse microscope featuring an automated stage interfaced to a
fluorescence light source and filter wheel and cubes with filters for
DAPI (excitation=350625 nm, emission=465625 nm) green
channel (excitation=490610 nm, emission=535615 nm), red
channel (excitation=555612.5 nm, emission=615626 nm), and
far-red (excitation=645615 nm, emission=715636 nm) chan-
nel fluorescence. The workstation includes a Windows computer,
which controls stage positioning, and data acquisition. Images
were acquired with a Hamamatsu Orca ERG progressive scan
134461024 cooled interline CCD camera, utilizing 2 x 2 binning.
Typically, 4 images (representing a 2 x 2 contiguous image set)
were acquired in the center of each well with either a 2060.5 NA
(resulting in 0.684860.6848 mm2//pixel) or a 4060.75 NA ‘‘dry’’
objective (resulting in 0.34460.344 mm2/pixel). Images were
stored as gray-scale bit mapped images (*.bmp).
An LSM 710 NLO Zeiss Multiphoton Laser Point Scanning
Confocal Microscope was used to scan adipocytes labeled with
newly developed phospho-perilipin 1A specific antibodies, and a
reference antibody to perilipin 1A. For these scans, a multi-photon
Mai-Tai DeepSee HP laser was used to excite DAPI with 728 nm
(pinhole=601 mm); single channel lasers at 514 (31 mm), 561
(36 mm), and 622 (42 mm) were used for image acquisitions in the
green, red, and far-red fluorescence channels, respectively.
2060.8 NA M27 Plan apochromat and 6361.4 NA M27 DIC
objectives were used.
High Content Analysis
CyteSeerH (Vala Sciences Inc) is a Java-based PC/Mac/Linux-
compatible cell image analysis program designed specifically for
high content analysis. CyteSeerH’s Colocalization Algorithm has
been specifically developed for the analysis of lipid droplets and
lipid droplet-associated proteins [20,24,25]. The Colocalization
Algorithm utilizes the nuclear image from each field of view to
identify the nuclei and to estimate cellular boundaries; this
information is then applied to the lipid and protein images, to
estimate the level of expression of lipid droplets and protein for
each cell. In this study, the data parameter Area Pm (area of the
protein mask), derived by the Colocalization Algorithm of
CyteSeerH is presented. Area Pm corresponds to the area within
each cell identified by the algorithm as being positive for the
expression of protein (mm2/cell). A second data parameter, Tii Pi
Pm, is reported for certain experiments in which the effect of
blocking peptides were tested for their ability to reduce the
labeling intensity obtained with the primary antibodies. Tii Pi Pm
is the ‘‘Total integrated intensity’’ (sum of intensities of the pixels),
in the ‘‘Protein image’’ (the image channel representing either
perilipin 1A or HSL), in the ‘‘Protein mask’’ (the pixels within the
cell, identified by CyteSeerH as being positive for protein
expression). Thus, Tii Pi Pm reports the overall intensity of the
cell for the protein that is labeled . In experiments in which
perilipin 1A and HSL were covisualized, the images were analyzed
twice, first for perilipin 1A, and second for HSL.
3T3L1 cells were plated and differentiated in 6 well plates. After
stimulation, cells in each well were rinsed with ice-cold PBS and
lysed in 75 ml buffer containing 50 mM Tris, pH 7.4, 100 mM
NaCl, 1% sodium deoxycholate, 4% Nonidet P-40, 0.4% SDS,
proteases and phosphatase inhibitors (Sigma). Lysates were
incubated on ice for 15 min, frozen overnight at 280uC, thawed
on ice and centrifuged at 13,000 rpm. The resulting supernatants
were stored at –80uC, and protein concentrations were determined
using the BCA kit (Pierce, Rockford, IL). Next, 5–40 mg of lysate
were loaded on 12% BisTris gel, subjected to electrophoresis and
transferred to BiotraceTM PVDF membrane (Pall, New York).
After blocking with 5% milk protein in TBST for 1 hour,
membranes were incubated overnight with primary antibodies.
Primary antibodies were detected using an HRP-conjugated
secondary antibody and visualized by ECL.
GraphPad Prizm was used to test for statistically significant
differences between controls and experimental groups (ANOVA
followed by Dunnett’s test, or ANOVA followed by Tukey’s test).
Generation of Monoclonal Antibodies Specific for
Phosphorylated Forms of Perilipin 1A
To generate novel monoclonal antibodies directed to phos-
phorylated forms of perilipin 1A, peptides analogous to the amino
acid sequences surrounding serine 497 (PKA-site 5) or serine 522
(PKA-site 6) of human perilipin 1A were synthesized. The serines
of these peptides were substituted with a chemical moiety (see
Materials and Methods), which acts as a mimetic for phospho-
serine. Mice were injected with the peptides, and sera-positive
animals were screened for MAb-secreting hybridomas. Aliquots of
media from the hybridomas were tested against the inoculating
peptides via ELISA. Clones that scored positive for recognition of
the inoculating peptides by ELISA were evaluated for their ability
to produce antibodies that visually label adipocytes pretreated with
forskolin in a pattern consistent with perilipin 1A. We selected two
monoclonal antibodies, designated anti-pPeri-site 5 and anti-
pPeri-site 6, that showed strong ELISA reactions and appropriate
Validation of the Specificity of the Antibodies
To evaluate the specificity of the anti-pPeri-site 5 and anti-
pPeri-site 6 antibodies, perilipin 1A mutant plasmids were utilized,
in which alanine was substituted for either serine 497 (to remove
Differential Phosphorylation of Perilipin 1A
PLOS ONE | www.plosone.org4 February 2013 | Volume 8 | Issue 2 | e55511
PKA-site 5) or serine 522 (to remove PKA-site 6). The coding
region of each perilipin 1A mutant construct was fused to
mCherry, a red channel fluorescent protein, to create mCh-
perilipin S497A and mCh-perilipin S522A. Separate HeLa cell
preparations were transiently transfected with GFP-perilipin 1A
(wt), mCh-perilipin 1A (S497A), or mCh-perilipin 1A (S522A).
The cells were then exposed to oleic acid to promote formation of
lipid droplets for 24 h. Subsequently, FSK+IBMX was used to
activate PKA. Cells were then fixed, stained for nuclei, and labeled
with either anti-pPeri-site 5 or anti-pPeri-site 6. Antibody labeling
was visualized with secondary anti-mouse antibodies conjugated to
a far-red fluorophore.
For cells prepared in this manner, the anti-pPeri-site 5 antibody
labeled cells expressing wild type perilipin 1A (Figure 2A), but did
not label cells expressing perilipin 1A with the S497A mutation
(Figure 2B). Similarly, the anti-pPeri-site 6 antibody also labeled
cells expressing wild type perilipin 1A (Figure 2C), but did not
label cells expressing perilipin 1A with the S522A mutation
(Figure 2D). These experiments demonstrate that specific labeling
of perilipin 1A by anti-pPeri-site 5 and anti-pPeri-site 6 requires
serine phosphorylation at position 497 and 522, respectively.
Guinea pig polyclonal antibody #GP29, raised against the N-
terminus of perilipin 1A, is widely used to label human  and
mouse 3T3L1 adipocytes . To further validate the labeling of
perilipin 1A by the anti-pPeri-site 5 and anti-pPeri-site 6
antibodies, 3T3L1 adipocytes were exposed to either 6 mM FSK
or 100 nM L-c-MSH for 20 minutes, then fixed, permeabilized,
and colabeled with either anti-pPeri-site 5 plus GP29, or with anti-
pPeri-site 6 plus GP29. This was possible since the murine and
guinea pig antibodies are from different host animals, and can thus
be specifically visualized with different secondary antibodies and
For images acquired from these FSK-treated adipocytes, via
conventional microscopy, the labeling by anti-pPeri-site 5 was
virtually identical to the labeling pattern obtained with GP29
(Figure 3A vs. 3B). Furthermore, virtually identical labeling
patterns were also seen for anti-pPeri-site 6 compared to GP29
(Figure 3C vs. 3D). Indeed, Pearsons’ Correlation Coefficients,
calculated on a cell by cell basis by CyteSeerH, yielded a value of
0.96 for the cell depicted in Figure 3A and 3B. For 3C and 3D,
there are two juxtaposed cells (note the two dark central regions in
3D, which correspond to the location of separate nuclei), for which
CyteSeerH calculated Pr values of 0.88 (cell 1) and 0.94 (cell 2),
respectively. Pr has a theoretical range from 21.0 (perfect
exclusion) to +1.0 (perfect coincidence). Thus, the observed Pr
values strongly support the observation from visual inspection that
distribution of phospho-perilipin 1A labeled by the monoclonal
antibodies coincides with the distribution of total perilipin 1A
labeled by GP29. For all of the antibodies, labeling was specific for
the edges of lipid droplets, which is the known intracellular
location of perilipin 1A. Virtually identical images for anti-pPeri-
site 5 and GP29 and for anti-pPeri-site 6 and GP29 were also
obtained by confocal microscopy (Figure 3E to 3H), confirming
the labeling at the edges of the lipid droplets by both anti-pPeri-site
5 and anti-pPeri-site 6, and the coincidence of the labeling with
In a further experiment to test the specificity of the antibodies,
human preadipocytes were transfected with either control siRNA
(scrambled sequence) or an siRNA corresponding to human
perilipin 1A, at concentrations ranging from 0 to 50 nM.
Following transfection, the cells were exposed to Differentiation
Medium for 6 days, treated with 6 mM FSK for 20 minutes, and
fixed and labeled for nuclei, lipid droplets, and for both phospho-
perilipin (utilizing either anti-pPeri-site 5 or anti-pPeri-site 6) and
total perilipin (utilizing GP29). For cells transfected with 10 nM
control siRNA, most of the cells featured numerous lipid droplets
and were strongly labeled by GP29, anti-pPeri-site 5, and anti-
pPeri-site 6 (Figure 4A and 4B). In contrast, cells transfected with
siRNA to perilipin 1A featured much weaker labeling by anti-
pPeri-site-5, anti-pPeri-site6, and GP29. Interestingly, the cells
transfected with the perilipin siRNA also featured fewer lipid
droplets. This is consistent with expectations, as knockout of
perilipin 1A in mice results in a leaner phenotype [27,28] and
siRNA to perilipin also downregulates lipid droplets in a murine
adipocyte cell model . The potential explanation for this is that
reduction of perilipin expression in adipocytes likely leads to
increased basal lipolysis and reduction of triglyceride stores.
To quantify the effects of the siRNAs, images from the
experiment were analyzed with CyteSeerH. Area Pm values were
not affected by the control siRNA, but the perilipin siRNA
reduced labeling by GP29, anti-pPeri-site5, and anti-pPeri-site 6 in
a very similar fashion, up to 90% (Figure 4C–E).
The above results confirm that anti-pPeri-site 5 labels perilipin
1A which has been phosphorylated at PKA-site 5, whereas anti-
pPeri-site 6 labels perilipin 1A, which has been phosphorylated at
PKA-site 6. Note that for the purposes of quantification, perilipin
1A labeled by the anti-phospho-perilipin 1A antibodies, will be
referred to as pPeri-site 5 and pPeri-site 6, respectively.
Labeling of Endogenous Phospho-perilipin 1A in Human
Adipocytes at the Initiation of Lipolysis
Next, experiments were conducted to test the ability of these
antibodies to recognize endogenous phospho-perilipin 1A in
adipocytes under lipolytic treatments. Human adipocytes exposed
to control medium showed little labeling for with the anti-pPeri-
site 5 antibody (Figure 5A and 5B). In contrast, following exposure
to FSK/IBMX, labeling by anti-pPeri-site 5 was very strongly
increased (Figure 5C and 5D), suggesting FSK/IBMX strongly
increases phosphorylation of perilipin 1A at PKA-site 5. Similarly,
for anti-pPeri-site 6, there was minimal labeling of human
adipocytes under basal conditions (Figure 5E and 5F), and
prominent labeling following exposure to FSK/IBMX (Figure 5G
and 5H), suggesting that FSK/IBMX also leads to phosphoryla-
tion of perilipin 1A PKA-site 6.
Differential Phosphorylation of Perilipin 1A in Response
to L-c-MSH Versus FSK
We  and others [18,19] have examined the phosphorylation
of HSL in 3T3L1 adipocytes in response to FSK and L-c-MSH.
To examine the phosphorylation of perilipin 1A in response to
these agents, murine 3T3L1 adipocytes were exposed to either
FSK or L-c-MSH for 5 minutes, then fixed, and labeled for nuclei
and lipid droplets, along with either anti-pPeri-site 5 or anti-pPeri-
site 6. 3T3L1 adipocytes exposed to control medium exhibited
relatively little labeling with anti-pPeri-site 5 (Figure 6A). Treating
the cells with FSK (6 mM) or L-c-MSH (100 nM) led to strong and
similar increases in labeling for anti-pPeri-site 5 (Figure 6B and
6C). Interestingly, the results obtained with the anti-pPeri-site 6
antibody were qualitatively distinct from the results for anti-pPeri-
site 5. For the anti-pPeri-site 6 antibody, labeling was dim for
control cells (Figure 6D) and increased by both FSK (Figure 6E)
and L-c-MSH (Figure 6F); however, the effect of L-c-MSH was
stronger than the effect of FSK.
To quantify the agonist-induced appearances of pPeri-site 5 and
pPeri-site 6, images from the experiment were analyzed with
CyteSeerH utilizing the Colocalization Algorithm. For pPeri-site 5,
Area Pm (the area of pixels that are positive for pPeri-site 5 on a
Differential Phosphorylation of Perilipin 1A
PLOS ONE | www.plosone.org5 February 2013 | Volume 8 | Issue 2 | e55511
per cell basis) averaged 27 mm2/cell for control cells (Figure 6G),
suggesting a small but measurable degree of basal phosphorylation
of perilipin 1A at PKA-site 5. Exposure to FSK or L-c-MSH led to
7-fold and 8-fold increases in Area Pm, respectively. These
responses were significantly different compared to control wells
(p,0.01), but were not significantly different from each other. For
pPeri-site 6, Area Pm values for controls averaged 7 mm2/cell
(Figure 6H). Exposure to FSK or L-c-MSH led to 11-fold and 27-
fold increases in Area Pm, respectively. Statistically, these
responses were different both from the controls and from each
other at a high level of significance (p,0.001). Thus, in this
experiment, L-c-MSH was nearly 3-fold more effective than FSK
at inducing the appearance of pPeri-site 6.
Concurrent Analysis of Phospho-perilipin 1A and
Phospho-HSL in 3T3L1 Adipocytes Subjected to FSK or L-
In the experiment described above, FSK and L-c-MSH were
equally effective at inducing the appearance of pPeri-site 5, but L-
c-MSH was a stronger agonist at inducing the appearance of
pPeri-site 6. This prompted us to ask whether or not these agents
might also lead to differential phosphorylation of HSL. Accord-
ingly, a time course experiment was conducted in which 3T3L1
adipocytes were exposed to control medium, 6 mM FSK, or
200 nM L-c-MSH, for time periods of 1, 5, or 20 minutes. The
cells were then fixed as before, but co-labeled for both phospho-
perilipin 1A and phospho-HSL. This was possible since the anti-
Figure 2. Determination of antibody specificity for perilipin 1A PKA-site 5, and PKA-site 6. Hela cells were transfected separately with
plasmids encoding wild-type (GFP, green) or mutant (mCherry, red) perilipin 1A (PLIN1) plasmids. Oleic acid was added for 24 h, followed by 7 min
treatment with 24 mM forskolin plus 125 mM IBMX and the cells labeled with either anti-pPeri-site 5 or anti-pPeri-site 6. For each condition, images are
shown for nuclei (DAPI), fluorescent protein (either GFP or m-Cherry), or for antibody labeling (the far red fluorescence channel). A, Cells expressing
GFP-w/t-perilipin 1A labeled with anti-pPeri-site 5. B, Cells expressing mCh-perilipin 1A S497A, which did not label for anti-pPeri-site 5. C, Cells
expressing GFP-w/t-perilipin 1A labeled with anti-pPeri-site 6. D, Cells expressing mCh-perilipin 1A S522A, which did not label with anti-pPeri-site 6.
Differential Phosphorylation of Perilipin 1A
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perilipin 1A antibodies were raised in mice, whereas the anti HSL
antibodies were raised in rabbit, and thus these antibodies are
immunologically distinct. In the experiments shown, adipocytes
were co-labeled with anti-pPeri-site 5 plus anti-pHSL-serine 563
antibodies, or co-labeled for with anti-pPeri-site 6 plus anti-pHSL-
serine 660 antibodies. Following labeling, the cells were visualized
in 4 fluorescent channels and the images analyzed with the
CyteSeerH Colocalization algorithm.
For pPeri-site 5 (Figure 7A), Area Pm values averaged about
20 mm2for control cells. Exposure to either FSK or L-c-MSH led
to approximately 4-fold increases in Area Pm which was
immediate and sustained throughout the time course. Thus,
phosphorylation of perilipin 1A at PKA-site 5 occurs rapidly for
both agonists, and, FSK and L-c-MSH were equally effective.
For pHSL-serine 563 (Figure 7B), Area Pm values for controls
were negligible. For cells exposed to FSK for 1 minute, Area Pm
for FSK was approx. 4 mm2whereas Area Pm for L-c-MSH
averaged about 20 mm2, which was significantly increased relative
to the controls. At the 5 minute time point, Area Pm values
averaged approx. 19 mm2for FSK and about 41 mm2for L-c-
MSH and these values were statistically different from controls. At
the 20 minute time point, the data for FSK and L-c-MSH were
somewhat higher than for the 5 minute time point. At all time
points, the differences between the L-c-MSH and FSK were
For pPeri-site 6 (Figure 7C) at the one minute time point, Area
Pm values averaged about 22 mm2for control samples, and FSK
and L-c-MSH increased Area Pm by approximate 20% and 50%,
respectively. At the 5 minute time point, the response for L-c-
MSH was 2-fold greater than the basal level of Area Pm, while the
effect of FSK was not significantly different from control cells. At
the 20 minute time point, FSK and L-c-MSH increased Area Pm
by approximately 3-fold and 5-fold, respectively. At all time points,
the data values obtained with L-c-MSH were significantly elevated
compared to the data for FSK.
For pHSL-serine 660 (Figure 7D), Area Pm values averaged
about 5 mm2for control cells at the 1 minute time point, whereas
FSK and L-c-MSH increased Area Pm by approximately 4-fold
and 9-fold, respectively. At the 5 minute time point, FSK and L-c-
MSH elicited similar responses and exhibited levels approximately
10-fold above the control values. At the 20 minute time point, both
agents elicited very strong, virtually identical responses.
It is worth emphasizing that the data for Figure 7A and 7B were
obtained from the same wells on the 96-well dish. Thus, FSK and
L-c-MSH elicited the appearance of pPeri-site 5 and pHSL-serine
563 with different kinetics and agonist efficacies within the same
samples. Similarly, the data for Figure 7C and 7D were also
obtained from the same wells on the 96-well dish, so FSK and L-c-
MSH also elicited the appearance of pPeri-site 6 and pHSL-serine
660 with different kinetics and different agonist efficacies within
the same samples. There are notable similarities in the data
obtained for pPeri-site 5 and pHSL-serine 660, including more
rapid phosphorylation of these sites, and equal efficacies of FSK
and L-c-MSH at the 5 and 20 minute time points, and similarities
between the data obtained for pPeri-site 6 and pHSL-serine 563
(phosphorylation of these sites occurred more slowly, and L-c-
MSH was a stronger agonist than FSK). Results with similar
kinetics and relative magnitudes of responses to FSK and L-c-
MSH were also obtained in two additional time course experi-
Differential Phosphorylation of Perilipin and HSL in
Response to a Panel of Lipolytic Agents
In related experiments, 3T3L1 cells were exposed to a panel of
lipolytic agents, which consisted of 0.01 mM isoproterenol, 0.1 mM
Figure 3. Labeling of adipocytes with anti-pPeri-site 5, anti-pPeri-site 6, and GP29. 3T3L1 adipocytes were exposed to agonists (either
6 mM FSK or 100 nM L-c-MSH) for 20 minutes, then fixed, and labeled with either anti-pPeri-site 5 or anti-pPeri-site 6 and GP29. Conventional
microscopy (A–D). Alexa FluorH 488 goat-anti-mouse and Alexa FluorH 647 goat anti-guinea pig secondary antibodies were used to visualize the
mouse monoclonal and guinea pig polyclonal antibodies, respectively. A, A FSK-treated cell visualized for anti-pPeri-site 5. B, The same cell, visualized
for GP29. C, A cluster of two FSK-treated cells (labeled 1 and 2) visualized for anti-pPeri site 6; the dashed line is the boundary between the cells as
estimated by CyteSeerH. D, The same cell cluster, visualized for GP29. Images were acquired with a 4060.75 NA objective. E–H, Confocal microscopy.
Alexa FluorH 568 goat-anti-mouse and Alexa FluorH 647 goat anti-guinea pig secondary antibodies were used to visualize the mouse monoclonal
and guinea pig polyclonal antibody labels, respectively. E, FSK-treated cells visualized for anti-pPeri-site 5. F, The same cells visualized for GP29.
Images were acquired with a 2060.8 NA objective. G, L-c-MSH-treated cells visualized for anti-pPeri-site 6. H, The same cells visualized for GP29.
Images were acquired with a 6361.4 NA objective. Examples of prominent labeling at the edges of lipid droplets are indicated by arrows (lipid
droplets, which were visualized in a separate channel, are not shown). Scale bars are 50 mm.
Differential Phosphorylation of Perilipin 1A
PLOS ONE | www.plosone.org7 February 2013 | Volume 8 | Issue 2 | e55511
isoproterenol, 1 mM FSK, 6 mM FSK, and 0.2 mM L-c-MSH, for
20 minutes, then fixed and labeled in a manner identical to the
experiments described above.
For pPeri-site 5 (Figure 8A), Area Pm for control cells averaged
approximately 70 mm2. The low and high concentrations of
isoproterenol increased Area Pm by approximately 30%, or 50%,
respectively. The results for both concentrations of FSK, and for
L-c-MSH were similar, with each agent increasing Area Pm by
For pHSL-serine 563 (Figure 8B), there was negligible labeling
of the control cells. The low and high concentrations of
isoproterenol increased Area Pm to approximately 20 mm2and
42 mm2; both of these values were significantly different from
controls and from each other. For FSK, the lower concentration
increased Area Pm to about 40 mm2whereas the high dose
increased Area Pm to about 80 mm2. L-c-MSH increased Area Pm
to approximately 103 mm2, which was the greatest observed effect
of the treatments, and the effect was significantly increased relative
to the data for the high dose of FSK.
For pPeri-site 6 (Figure 8C), Area Pm averaged 25 mm2for
control cells. Exposure to the low and high concentrations of
isoproterenol increased Area Pm by 2-fold and 3-fold, respectively.
The low and high concentrations of FSK elicited approximately
2.5-fold and 3-fold increases in Area Pm. L-c-MSH increased
Area Pm approx. 5-fold, which was the strongest response
observed in these experiments.
For pHSL-serine 660 (Figure 8D), Area Pm averaged 48 mm2
for controls. Exposure to the low and high doses of isoproterenol
increased Area Pm by 70% and 2-fold, respectively. For FSK, the
low and high concentrations elicited 3.8-fold and 4.3-fold increases
in Area Pm, whereas L-c-MSH increased Area PM by 4.2-fold.
In the above experiment, the response patterns obtained for
pPeri-site 5 and pHSL-serine 660 are strikingly similar with perfect
agreement in the rank order effectiveness of the treatments. The
response patterns for pPeri-site 6 and pHSL-serine 563 are also
very similar to each other and are different from the response
patterns for pPeri-site 5 and pHSL-serine 660.
Figure 4. Downregulation of anti-pPeri-site 5, anti-pPeri-site 6, and GP29 labeling by siRNA to perilipin 1A. Preadipocytes were
transfected with either control or perilipin 1A siRNA (0 to 50 nM), exposed to differentiation medium for 6 days, treated with 6 mM FSK for 20
minutes, then fixed and labeled for nuclei (blue), lipid (green), and either anti-pPeri-site 5 or anti-pPeri-site 6 (red) plus GP29 (yellow). A,
Representative fields of view are shown for cells transfected with 10 nM siRNA and labeled with anti-pPeri-site 5 plus GP29. B, Representative fields of
view are shown for cells transfected with 10 nM siRNA and labeled with anti-pPeri-site 6 plus GP29. C, D, and E are mean values for Area of the
Protein mask (Area Pm), for GP29, anti-pPeri-site 5, and anti-pPeri-site 6, respectively. Data are normalized to the 0 siRNA control, and each bar
represents the mean 6 SD, for n=6 wells. *** p,0.001 for perilipin vs. control siRNA at each concentration (Student’s t-test).
Differential Phosphorylation of Perilipin 1A
PLOS ONE | www.plosone.org8 February 2013 | Volume 8 | Issue 2 | e55511
Experiments with Blocking Peptides
The close similarities between the results for pPeri-site 5 and for
pHSL-serine660, and the similarity between the results for pPeri-
site 6 and pHSL-serine 563 raised the possibility that the
antibodies to phospho-perilipin may be labeling phospho-HSL,
or vice versa. To test if these peptides would block labeling of the
cells by the primary antibodies, cells were incubated with primary
antibodies that were pre-incubated with blocking peptides
corresponding to pPeri site 5, pPeri site 6, or pHSL serine 660.
For 3T3L1 adipocytes treated with 1 mM isoproterenol,
preincubation with the pPeri site 5 peptide reduced the overall
image intensities resulting from labeling by the anti-pPeri site 5
antibody, and the Tii Pi Pm data parameter (which represents the
protein labeling intensity, quantified on a per cell basis) (Figure 9A).
Thus, binding of anti-pPeri site 5 antibody to its target is blocked
by peptide corresponding to pPeri site 5, confirming the specificity
of the antibody. In contrast, the pPeri site 5 peptide did not reduce
image intensities or Tii Pi Pm resulting from labeling by the anti-
pHSL serine 660 antibody (Figure 9A); thus, the anti-pHSL serine
660 antibody does not bind to pPeri site 5.
In a reciprocal experiment, also featuring 3T3L1 adipocytes
treated with 1 mM isoproterenol, preincubation of anti-pPeri-site 5
antibody with the pHSLser660 peptide did not reduce overall
image intensities or Tii Pi Pm obtained with the anti-pPeri-site 5
antibody (Figure 9B); in contrast, the pHSL serine 660 peptide
strongly reduced the image intensities and Tii Pi Pm values
obtained with the anti-pHSL-serine 660 antibody (Figure 9B). The
results demonstrate that the anti-pPeri-site 5 antibody likely does
not bind to pHSL serine 660 and confirms the specificity of the
anti-pHSL-serine 660 antibody.
Finally, for 3T3L1 adipocytes treated with 100 nM L-c-MSH,
preincubation of anti-pPeri-site 6 antibody with a peptide
corresponding to pPeri-site 6 strongly reduced image intensities
and the Tii Pi Pm values obtained with anti-pPeri-site 6 antibody,
but did not reduce overall image intensities or the Tii Pi Pm values
obtained with the anti-pHSL-serine 563 antibody (Figure 9C). The
results confirm the specificity of the anti-pPeri-site 6 antibody for
pPeri-site 6, and demonstrate that the anti-pHSL-serine 563
antibody does not bind to pPeri-site 6.
Overall, the results with the blocking peptides confirmed the
specificity of the phospho-perilipin and phospho-HSL antibodies
for labeling their intended targets and indicate it is highly unlikely
that the anti-perilipin 1A antibodies recognize HSL, or vice versa.
Analysis of Perilipin 1A Phosphorylation via Western
To explore additional applications for the monoclonal antibod-
ies against phospho-perilipin, we tested the ability of these
antibodies to identify phospho-perilipin 1A by Western blotting.
In our hands, anti-pPeri-site 5 did not visualize specific bands on
Western blots. In contrast, anti-pPeri-site 6 labeled a single band,
which migrated at approx. 60 KDa, which is consistent with the
molecular weight of perilipin 1A, and the intensity of this band was
strongly increased in samples for 3T3L1 cells treated with forskolin
(Figure 10A). Furthermore, for experiments in which adipocytes
were treated for 5 minutes with control, 6 mM FSK, 1 mM
isoproterenol, and 100 nM L-c-MSH, the bands from cells treated
with isoproterenol and L-c-MSH recognized by the anti-pPeri-site
6 antibody were more intense than the band for cells treated with
FSK (Figure 10B). The observation is consistent with the previous
observations that L-c-MSH is a stronger agonist than FSK for
phosphorylation of perilipin at PKA-site 6, as quantified via the
microscopy methods. The strong response to isoproterenol, vs.
FSK is somewhat unexpected considering the results from the
microscopy assay. However, in the experiment for the Western
blot, the cells were treated with a higher concentration of
isoproterenol than utilized in the microscopy experiment. Results
similar to those shown in Figure 10 were obtained in 2 additional
experiments with anti-pPeri-site 6. In all, the results confirm the
Figure 5. Labeling of FSK-treated human adipocytes with anti-pPeri-site 5 and anti-pPeri-site 6 antibodies. Human subcutaneous
adipocytes were exposed to either control medium, or medium supplemented with 10 mM FSK and 500 mM IBMX for 2 minutes, then fixed and
labeled for nuclei, lipid droplets, and with either anti-pPeri-site 5 or anti-pPeri-site 6. A goat-anti-mouse secondary antibody coupled to Texas Red
was used to visualize the phospho-perilipin antibodies in the red fluorescent channel. A, Control adipocytes are shown visualized for nuclei (blue) and
lipid droplets (green). B, the same field is shown visualized for anti-pPeri-site 5 (red). C, FSK/IBMX-treated adipocytes are shown visualized for nuclei
and lipid droplets. D, The same field is shown visualized for anti-pPeri-site 5. E, Control adipocytes are shown visualized for nuclei and lipid droplets. F,
The same field is shown visualized for anti-pPeri-site 6. G, FSK/IBMX-treated adipocytes are shown visualized for nuclei and lipid droplets. H, The same
field is shown, visualized for anti-pPeri-site 6. Scale bar=50 mm.
Differential Phosphorylation of Perilipin 1A
PLOS ONE | www.plosone.org9 February 2013 | Volume 8 | Issue 2 | e55511
anti-pPeri-site 6 antibody recognizes phosphorylated perilipin 1A
and this antibody is compatible with Western blotting.
The goal of this study was to examine the phosphorylation of
perilipin 1A for comparison to HSL phosphorylation, at the
initiation of lipolysis. Towards this purpose, we developed novel
monoclonal antibodies (anti-pPeri-site 5 and anti-pPeri-site 6),
which specifically recognize perilipin 1A phosphorylated at either
PKA site 5 or PKA site 6, respectively. The specificity of these
antibodies was confirmed in several ways. First, these antibodies
recognized the synthetic phospho-peptides corresponding to the
perilipin 1A PKA phosphorylation sites used in immunizing the
mice, by ELISA. In subsequent experiments, these antibodies
labeled ectoptically expressed perilipin 1A in FSK/IBMX-treated
HeLa cells with no labeling of other proteins endogenous to these
cells. Also, the labeling of ectopic perilipin 1A in HeLa cells was
eliminated by single point mutations in perilipin where alanine was
substituted for serine at the target site. The labeling by each
antibody was also increased by FSK, an agent known to stimulate
PKA-mediated phosphorylation of perilipin. The anti-pPeri-site 5
and anti-pPeri-site 6 antibodies labeled the edges of lipid droplets,
consistent with the cellular localization pattern for perilipin 1A,
and the labeling patterns were coincident with labeling by GP29, a
well-established guinea pig polyclonal antibody to perilipin 1A.
Finally, for FSK-treated human adipocytes, an siRNA specific for
Figure 6. Differential phosphorylation of perilipin 1A by forskolin (FSK) and LcMSH. 3T3L1 adipocytes were exposed to either FSK (6 mM)
or L-c-MSH (100 nM) for 5 minutes, then fixed and labeled for nuclei (blue), lipid droplets (green), and phosphorylated perilipin (red). The cells were
then imaged (20Xobjective, 4 images/well) and the images analyzed utilizing the Colocalization algorithm. A, B, and C, Images are shown of
adipocytes exposed to control, FSK, or LcMSH, respectively, labeled with anti-pPeri-site 5. D, E, and F, Images are shown of adipocytes exposed to
control, FSK, LcMSH, respectively, labeled with anti-pPeri-site 6. G and H represent Area Pm for pPeri-site 5 and for pPeri-site 6, respectively. Each bar
represents the mean SD for n=3 wells/condition (an average of 532 cells/well) for G, or n=8 well/condition (an average of 272 cells/well) for H. **
p,0.01 vs. Con (ANOVA followed by Tukey’s test). *** P,0.001 vs. Con.###p,0.001 vs. FSK. Scale bar=50 mm.
Differential Phosphorylation of Perilipin 1A
PLOS ONE | www.plosone.org10 February 2013 | Volume 8 | Issue 2 | e55511
perilipin 1A down-regulated labeling by anti-pPeri-site 5, anti-
pPeri-site 6, and GP29 in an identical manner. As pPeri-site 5 and
pPeri-site 6 are associated with the carboxyl terminal of the
protein, the antibodies are specific perilipin 1A, which is the full-
length form of perilipin . Notably, anti-pPeri-site 6 has recently
been utilized by others to characterize the control of lipolysis in a
variety of contexts; findings from these studies include increased
lipolysis and phosphorylation of perilipin 1A associated with
adaptive thermogenesis , increased lipolysis and phosphoryla-
tion of perilipin 1A on downregulation of the Berardinelli-Seip
Congenital Lipodrystrophy 2/Seipin (BSCL2) protein ,
diminished b-adrenergic-induced lipolysis and phosphorylation
of perilipin 1A on downregulation of CD36 , and increased
lipolysis with unchanged phosphorylation of perilipin 1A in
response to Fas .
Our results demonstrate that both perilipin 1A and HSL have
sites that are phosphorylated at different rates in response to
lipolytic agents and these sites are phosphorylated in response to
lipolytic agents, in a strikingly similar manner. For example,
perilipin 1A PKA-site 5 and HSL-serine 660 were phosphorylated
rapidly and with equal efficacy in 3T3L1 adipocytes exposed to
either FSK or L-c-MSH. In contrast, perilipin 1A PKA-site 6 and
HSL serine 563 were phosphorylated more slowly, and L-c-MSH
was a markedly stronger agonist for this effect than FSK. Indeed,
when cells were exposed to an extended panel of lipolytic agents,
the pattern of results for pPeri-site 5 was virtually identical to that
for pHSL-serine 660, whereas the pattern of results for pPeri-site 6
were very similar to that for pHSL-serine 563. These similarities
were not due to recognition of HSL by the antibodies raised to
perilipin 1A or recognition of perilipin 1A by the antibodies raised
to HSL, as the antibodies were selectively blocked by peptides that
correspond to their intended targets.
The differential phosphorylation of HSL (more rapid at serine
660 vs. serine 563 in response to FSK) was first observed by
Martin and colleagues , who hypothesized this might be
accomplished by separate pools of PKA anchored to different
cellular locations (e. g., the cytoplasmic side of the plasma
membrane, or the edges of the lipid droplets). While our data is in
agreement with the likely role of PKA-anchoring proteins such as
Optic Atrophy-1  in orchestrating the phosphorylation of
perilipin 1A and HSL, an important distinction is perilipin 1A
remains associated with lipid droplets during the initiation of
Figure 7. Phosphorylation of Perilipin 1A and HSL in response to FSK and L-c-MSH. 3T3L1 adipocytes were exposed to control medium,
6 mM forskolin (F), or 200 nM L-c-MSH (M) for 1, 5, or 20 minutes, then fixed and co-labeled with either anti-pPeri-site 5+ anti-pHSL-serine 563, or anti-
pPeri-site 6+ anti-pHSL-serine 660. Images were obtained with a 20X objective (4 images/well, representing an average of 930 cells/well). A and B
represent Area Pm values obtained for pPeri-site 5 and pHSL-serine 563. C and D represent Area Pm values obtained for pPeri-site 6 and pHSL-serine
660, respectively. Each bar represents the mean 6 SD for n=7 to 8 wells. ** p,0.01 compared to controls (ANOVA followed by Tukey’s test); ***
p,0.001 compared to controls.ap,0.05 vs. controls and M;bp,0.05 vs. controls and F.
Differential Phosphorylation of Perilipin 1A
PLOS ONE | www.plosone.org11 February 2013 | Volume 8 | Issue 2 | e55511
lipolysis, whereas HSL undergoes a translocation step. Thus,
perilipin 1A is differentially phosphorylated in a manner similar to
HSL, even though perilipin 1A remains stationary. Since perilipin
1A is stationary, the differential phosphorylation of perilipin 1A is
unlikely due to pools of PKA sequestered in different cellular
locations by PKA-anchoring proteins, as suggested for HSL .
Consideration of the amino acid sequences of the PKA
phosphorylation sites on perilipin 1A and HSL leads us to suggest
an alternative hypothesis to account for the differential phosphor-
ylation of these proteins. The consensus sequence for PKA is
considered to be R(R/K)X(S/T) . While the sequences
surrounding perilipin 1A PKA site 5, perilipin 1A PKA site 6,
and HSL serine 660 conform to this sequence, the sequence
surrounding HSL-serine 563 does not, as there is no arginine at
the P-3 position (Figure 1B). Even for phosphorylation sites that
conform to the consensus sequence, it is well known that different
sequences are phosphorylated more readily by PKA than others.
For example, peptides with arginines at both the P-2 and P-3
Figure 8. Differential phosphorylation of perilipin 1A and HSL in response to a panel of lipolytic agents. 3T3L1 adipocytes were
exposed to the indicated concentrations (mM) of isoproterenol (ISO), FSK, and L-c-MSH for 20 minutes, then fixed and labeled either PKA-site 5+ pHSL-
serine 563 or PKA-site 6+ pHSL-serine 660. The cells were imaged with a 20X objective, 4 images/well, yielding an average of 360 cells/well. Area Pm
values are shown for (A) PKA-site 5 (B), pHSL-serine 563, (C) PKA-site 6, and (D), pHSL-serine 660, respectively. Each bar represents the mean 6 SD for
n=8 wells. * p,0.05 vs. control (ANOVA followed by Tukey’s test procedure); *** P,0.001 vs. controls. Within each panel, conditions distinct from
one another at the p,0.05 level are also designated (a,b,c,d).
Differential Phosphorylation of Perilipin 1A
PLOS ONE | www.plosone.org12 February 2013 | Volume 8 | Issue 2 | e55511
positions (such as perilipin PKA site 5 and HSL serine 660) have a
higher affinity for PKA than peptides containing only a single
arginine at either position, (such as perilipin PKA site 5 and HSL
serine 563) [36,37,38]. Surveys of the human genome suggest 80%
of sequences represented by RRXS are physiological substrates for
PKA. In contrast, only 48% of sequences represented by RKXS
are PKA substrates, likely reflecting the lower affinity of the
RKXS sequences for PKA [39,40]. The sequences shown for
perilipin 1A PKA sites 5 and 6, and for HSL serine 563 and serine
660 are conserved across a variety of mammals, including human,
mouse, rat, and pig. We hypothesize perilipin PKA 1 site 5 and
HSL-serine 660 have a higher affinity for PKA, and are thus
phosphorylated more readily than perilipin 1A PKA site 6 and
HSL-serine 563, due to the presence of arginines in both the P-2
and P-3 positions of perilipin 1A PKA site 5 and HSL-serine 660.
The fact that such sequences are evolutionarily conserved suggests
both high- and low-affinity sites for PKA phosphorylation are
likely required for the functions of perilipin 1A and HSL.
Indeed, perilipin 1A PKA-site 5 and site 6 have previously been
linked to different physiological processes (lipid droplet-dispersion
for PKA site 5 , and ATGL-dependent lipolysis for PKA site 6
). It is intriguing that lipid droplet dispersal, which requires
several hours to manifest, is regulated by phosphorylation of
perilipin 1A PKA-site 5, a site that is rapidly phosphorylated.
Furthermore, the matching results for phosphorylation of perilipin
1A PKA-site 5 and HSL-serine 660 by the panel of lipolytic agents
suggest that these phosphorylation events may be related, but HSL
has not been implicated in lipid droplet dispersal. The results
emphasize that much remains to be elucidated about the
relationships between phosphorylation of perilipin 1A and HSL
and the orchestration of the lipolytic response.
The reason for the greater effectiveness of L-c-MSH compared
to FSK at inducing the appearance of perilipin 1A PKA-site 6 and
pHSL-serine 563 is unclear. One possibility is these sites are
Figure 9. Tests for antibody specificity utilizing blocking peptides. 3T3L1 adipocytes were exposed to either 1 mM isoproterenol or 100 nM
L-c-MSH. Prior to labeling, primary antibodies were preincubated with the indicated amounts (mg) of blocking peptides corresponding to pPeri-site 5,
pPeri-site 6, or pHSL-serine 660. Anti-phospho-perilipin 1A and anti-phospho-HSL antibodies were visualized in the red and far-red fluorescence
channels, respectively. A, Results are shown for cells treated with isoproterenol for 15 minutes in which anti-pPeri-site 5 and anti-pHSLserine 660 were
blocked with pPeri-site 5. Upper panels depict cell images. Lower bar graphs depict Tii Pi Pm data for pPeri-site 5 and pHSL-serine 660. B, Results are
shown for cells treated with isoproterenol for 10 minutes in which anti-pPeri-site 5 and anti-pHSL-serine 660 were blocked with pHSL-serine 660. C,
Results are shown cells treated with L-c-MSH for 7 minutes in which anti-pPeri-site 6 and anti-pHSLserine563 were blocked with pPeri-site 6. For A,
each bar represents a single well; for B and C, each bar represents the mean 6 SD for n=3 wells.
Figure 10. Phospho-perilipin 1A visualized via Western blot-
ting. 3T3L1 adipocytes were exposed to lipolytic agonist for 5 minutes;
whole cell lysates were then prepared and subjected to SDS-PAGE/
Western blotting (20 mg protein/lane) utilizing the anti-pPeri-site 6
antibody. A, Results are shown for cells exposed to either control
medium (C) or 6 mM FSK (F). B, Results are shown for a separate
experiment in which cells were exposed to control medium, 6 mM FSK,
1 mM isoproterenol (I), or 100 nM L-c-MSH (M).
Differential Phosphorylation of Perilipin 1A
PLOS ONE | www.plosone.org 13 February 2013 | Volume 8 | Issue 2 | e55511
substrates for an additional kinase, other than PKA, which is
activated by L-c-MSH. Cyclic-GMP-dependent Protein Kinase
(PKG), might be a candidate for this as it shares substrate
specificity with PKA and is present in 3T3L1 adipocytes .
Furthermore, atrial natriuretic peptide likely activates lipolysis in
human adipocytes by PKG-mediated phosphorylation of HSL at
serine 660 . However, L-c-MSH is not known to activate
High content analysis methods provide several advantages over
traditional immunofluorescence labeling and analysis techniques.
The high throughput plating configuration enables processing of a
larger number of samples per experiment than is practical with
procedures in which cells are plated on coverslips, which must be
handled individually and mounted on slides. Secondly, the image
analysis algorithms objectively analyze each cell imaged, providing
quantitative data for hundreds of cells per condition. High content
analysis permits discrimination between effects subtly different
between experimental treatments and greater resolving power for
statistical analysis. While high content analysis strategies have most
commonly been utilized in high throughput screening applica-
tions, our study demonstrates the value of utilizing HCA to
examine the early events associated with activation of lipolysis, a
pathway of key importance to obesity and the metabolic
syndrome. Our data suggests future directions for research such
as testing the hypothesis that differential phosphorylation of
perilipin 1A and HSL depends upon the amino acid sequences
flanking the phosphorylation sites, and to pursue further elucida-
tion of the mechanisms by which L-c-MSH elicits phosphorylation
of perilipin 1A and HSL.
We wish to thank Dr. Andrew Bicknell for generously providing us with
Lys-c3-MSH. Excellent technical assistance was also provided by Denise
Carroll, and Ed Monosov (Sanford-Burnham Medical Research Institute).
The anti-pPeri-site 5 and anti-pPeri-site 6 monoclonal antibodies were
produced by the Monoclonal Antibody/Baculovirus Shared Resource of
the Dan L. Duncan Cancer Center at Baylor College of Medicine.
Conceived and designed the experiments: DM-L SMH RW AH BMB
MAM JHP PMM. Performed the experiments: DM-L SMH RW AH JBN
RAH KC PMM. Analyzed the data: SMH RW MAM JHP PMM.
Contributed reagents/materials/analysis tools: JBN BMB MGM DPE,
JHP. Wrote the paper: DM-L SMH PMM.
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