Journal of Biomolecular Screening
The online version of this article can be found at:
2006 11: 269J Biomol Screen
Yuhong Du, Shane C. Masters, Fadlo R. Khuri and Haian Fu
Monitoring 14-3-3 Protein Interactions with a Homogeneous Fluorescence Polarization Assay
On behalf of:
Journal of Biomolecular Screening
can be found at:
Journal of Biomolecular Screening
Additional services and information for
What is This?
- May 5, 2006 Version of Record >>
by guest on October 31, 2013 by guest on October 31, 2013 by guest on October 31, 2013 by guest on October 31, 2013 by guest on October 31, 2013 by guest on October 31, 2013 by guest on October 31, 2013 by guest on October 31, 2013 by guest on October 31, 2013jbx.sagepub.comjbx.sagepub.comjbx.sagepub.com jbx.sagepub.com jbx.sagepub.comjbx.sagepub.com jbx.sagepub.comjbx.sagepub.comjbx.sagepub.com Downloaded from Downloaded from Downloaded from Downloaded from Downloaded from Downloaded from Downloaded from Downloaded from Downloaded from
10.1177/1087057105284862 ARTICLEDu et al. An HTSAssay for 14-3-3 Proteins
Monitoring 14-3-3 Protein Interactions with a Homogeneous
Fluorescence Polarization Assay
1SHANE C. MASTERS,
1,2FADLO R. KHURI,
3and HAIAN FU
The 14-3-3 proteins mediate phosphorylation-dependent protein-protein interactions. Through binding to numerous client
cancer and neurodegenerative disorders. To better understand the structure and function of 14-3-3 proteins and to develop
small-molecule modulators of 14-3-3 proteins for physiological studies and potential therapeutic interventions, the authors
have designed and optimized a highly sensitive fluorescence polarization (FP)–based 14-3-3 assay. Using the interaction of
valuable for high-throughput screening of 14-3-3 modulators. (Journal of Biomolecular Screening 2006:269-276)
14-3-3, fluorescence polarization, protein-protein interaction
(pS/T)–containing motifs used by a variety of signal transduction
pathways.1-7To date, 14-3-3 proteins have been reported to bind
3 proteins play important roles in a wide range of vital regulatory
processes, such as Bad-induced apoptosis, Raf-1-mediated cell
pathological conditions, such as neurodegenerative disorders and
cancers.11-13Thus, such studies on the 14-3-3/client-protein inter-
actions may provide tremendous opportunities for therapeutic in-
terventions. However, no chemical tools are available that allow
pharmacological probing of 14-3-3 function under in vivo
HE 14-3-3 PROTEINS ARE THE PROTOTYPE for a novel class of
protein modules that can recognize phosphoserine/threonine
high-affinity peptide, termed R18, has been identified and well
studied. The specificity of R18 for 14-3-3 has been confirmed by
an affinity pull-down assay, mutational analysis, and
cocrystallization analysis.14-16Functionally, R18 and its derivative
difopein can specifically disrupt the interaction of 14-3-3 with a
broad range of cellular proteins, such as Raf-1.14,15R18 and difo-
protein interactions in various biological systems and in
and resistance of cells to cytotoxic agents, as shown by the ability
of R18 to enhance cell death induced by anticancer drugs such as
cisplatin.15Other examples of peptide 14-3-3 antagonists include
those derived from natural 14-3-3 binding proteins. One such an-
tagonist is the penetratin-linked AARAApSAPA peptide (AP-
pSAPA).17AP-pSAPA, when transduced into cells, blocked the
IGF-1-induced binding of 6-phosphofructo-2-kinase to 14-3-3.
However, delivery of a peptide inhibitor into cells or animals and
the instability of these peptides in vivo pose significant obstacles
for mechanistic and therapeutic exploration.
cilitate such work. Although protein-protein interactions are gen-
erally difficult to disrupt, recent studies have yielded some suc-
cesses, such as small-molecule inhibitors for the MDM2/p53
interaction and the Smac/XIAP association.18-20In support of the
strategy to target 14-3-3 proteins with a small molecule, a single
© 2006 Society for Biomolecular Scienceswww.sbsonline.org269
Chemistry-Biology Discovery Center, Emory University, Atlanta, GA.
2Current address: Medical College of Georgia, Augusta, GA.
3Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA.
tion Nov 15, 2005.
Journal of Biomolecular Screening 11(3); 2006
charge-reversal mutation, K49 to E, in the amphipathic groove of
14-3-3 is sufficient to disrupt the 14-3-3/client-protein interac-
rupt the 14-3-3/client-protein interaction with a small molecule.
3 protein interactions. The FP method measures increased polar-
ization of emitted lightin response to decreased rotation of the la-
beled molecule, for example, through protein complex formation
tion of 14-3-3 with a rhodamine-labeled peptide derived from a
riety of client proteins. Because of its simplicity, it is particularly
suited for high-throughput screening (HTS) to isolate small-
demonstrated signal-to-noise ratio greater than 10 and a Z′ factor
greater than 0.7. Such a validated assay can be readily used for
screening large chemicalcompound libraries in an HTS formatto
its protein partners.
MATERIALS AND METHODS
All of the reagents were of highest purity and were obtained
from Sigma Chemical Co. (St. Louis, MO), unless otherwise as
Fluorescent peptide probe and peptide antagonists
1 was synthesized and labeled with 5/6 carboxytetramethyl-
contains 15 residues: 5/6-TMR-LSQRQRST[pS]TPNVHM,
low). The following peptides were synthesized and purified at the
Emory Microchemistry and Proteomics Facility: Biotin-pS259-
Raf, pS967-ASK1 (GSNEYLKSI[pS]LPVP29), R18
(PHCVPKNLSWLNLEAN MCLP14), and the mutated R18,
R18Lys, where D12 and E14 were changed to K.15
Expression and purification of recombinant 14-3-3 proteins
The recombinant GST-14-3-3γ protein was expressed in Esch-
erichia coli strain BL21 (DE3) as a GST-tagged product as de-
scribed.30E. coli BL21 (DE3) strains were grown in LB medium
with ampicillin (100 µg/mL) for protein auto-induction. After
overnight at 37 °C, the cells were harvested and the pellet was re-
suspended in ice-cold phosphate-buffered saline (PBS) buffer
composed of phenylmethylsulfonyl fluoride (PMSF, 1.0 mM),
leupetin (1 µg/mL), and aprotinin (1 µg/mL). The GST-14-3-3γ
protein was then purified by using Glutathione-Sepharose beads
(GE Healthcare, Piscaraway, NJ), and the bound protein was
eluted in a PBS buffer (pH 9) containing reduced glutathione (20
mM), PMSF (0.25 mM), dithiothreitol (DTT; 5 mM), and Triton
X100 (0.1%). Salts were removed from the pooled elution frac-
7.4). The protein concentration was measured using the Bradford
method (BioRad), and GST-14-3-3γ was stored at –20 °C before
use. The recombinant hexaHis-tagged 14-3-3 isoforms were ex-
pressed and purified essentially as previously described.31
Fluorescence polarization measurements
The reaction buffer used in the FP assay throughout this study
contains HEPES (10 mM, pH 7.4), NaCl (150 mM), Tween-20
(0.05%), and DTT (0.5 mM). FP measurements were performed
using black 384-well microplates (Corning Costar, Cambridge,
MA) on an Analyst HT plate reader (Molecular Devices,
Sunnyvale, CA). An integration time of 100 ms was used, and Z
height was set at 2.15 mm (middle). The excitation polarization
nm. All fluorescence polarization values were expressed as the
millipolarization (mP) units.28The assay window is calculated as
and the FP value recorded for the free peptide probe.
Assay development and optimization
The 14-3-3 FP assay was carried out in black 384-well
For each assay, 2 basic solutions were prepared. Solution A con-
with 2 nM of the peptide probe or as specified). Solution B con-
of 14-3-3 or serial dilutions of a specified concentration, as de-
scribed in the legends to the figures, or without 14-3-3 proteins as
lution A and solution B was mixed and incubated for 60 min at
room temperature (RT) or a defined period of time. The polariza-
For assay performance, the signal-to-noise ratio (S:N) and the
ations for bound (b) and free (f) peptides without 14-3-3, whereas
µb– µfis the difference in mean signals for bound and free pep-
Excel (Microsoft Corporation, Redmond, WA) and Prism 4.0
(Graphpad Software, San Diego, CA).
Du et al.
270www.sbsonline.orgJournal of Biomolecular Screening 11(3); 2006
Competitive FP assay
in a 384-well format with TMR-pS259-Raf (1 nM), GST-14-3-3γ
(0.5 µM, or as specified), and serial dilutions of competitive pep-
pS259-Raf peptide only (free peptide) and TMR-pS259-Raf pep-
tide with GST-14-3-3γ (bound peptide) were included as controls
in each plate.Thepolarization valuesweremeasured after60 min
of incubation at RT. The competitive effect was expressed as the
with nonlinear regression analysis.
To evaluate the quality or suitability of the FP assay for HTS,
the assay windows, the Z′ factors, and the S:N ratios were deter-
mined from assays carried out in 40 microplates (384-well for-
384-well plate contained 8 wells of free TMR-pS259-Raf peptide
control and 8 wells of bound TMR-pS259-Raf peptide control
(with GST-14-3-3γ, 0.5 µM). ThemPvaluefor each wellwasob-
tained and analyzed, and means of both free and bound peptide
ter plot. The Z′ factor and the S:N ratios were calculated as
RESULTS AND DISCUSSION
Development of 14-3-3 protein fluorescence polarization assay
FP is a very powerful and sensitive nonradioactive technique
can be used to measure binding and dissociation between 2 mole-
cules if 1 of the binding molecules is relatively small and fluores-
cent. When fluorescent small molecules (such as a small peptide)
in solution are bound to bigger molecules (such as a protein), the
is irradiated with polarized light, much of the emitted light is also
change in polarization. We reasoned that this assay format might
be particularly suited to the study of 14-3-3 proteins because the
erally mediated by well-defined short peptide motifs in the client
labeled for FP assay development.
terminalsequencein ligand binding (seesupplementary materials
ground signal. To develop a robust FP assay with reduced back-
ground polarization using a natural client protein, we used the
Raf peptide (2 nM in a 2× solution) was mixed in a 1:1 ratio with
various concentrations of GST-14-3-3γ solution (2×), and FP was
measured. The initial concentration of the TMR-pS259-Raf was
chosen based on the observation that 1 nM of the TMR-labeled
parallel channel than the “buffer-only” control samples. Interac-
tion of 14-3-3 with TMR-pS259-Raf gave rise to a significant FP
signal with a minimal background polarization with the peptide
probe alone (Fig. 1). As shown in Figure 1A, with increasing
amounts of GST-14-3-3γ protein, polarization values progres-
tion of fluorescent peptide was bound to the 14-3-3 protein. The
maximum assay window (∆mP = mP of bound peptide – mP of
free peptide) reached approximately 150 mP with an estimated
dissociation constant, Kd, of 0.412 ± 0.01 µM for the Raf peptide
TMR-pS259-Raf with 14-3-3γ because TMR-pS259-Raf with
(Fig. 1A) and because the signal can be competed away by
tative “mix-and-measure” assay for studying 14-3-3 protein
say that is simple for routine use and highly robust for HTS. We
therefore optimized the performance of the assay and assessed its
termined based on concentration-dependent interactions of 14-3-
sensitive to changes in 14-3-3 concentration up to about 0.5 µM.
1C). The S:N value is significantly enhanced upon increasing the
3-3 is used in the reaction and the ratio continues to improve to
reach greater than 25. The Z′ factor incorporates the dynamic
The Z′ factor improves as the concentration of 14-3-3γ increases
than 10 when 14-3-3γ concentration is greater than 0.1 µM, indi-
teins was used in the subsequent FP experiments.
To examine whether the GST tag has any effect on the 14-3-3
FP assay, a hexaHis-tagged 14-3-3γ protein was used in the same
lar interaction profile with an almost identical Kd value (0.42 ±
say for 14-3-3 isoforms, we tested additional hexaHis-14-3-3
isoforms. Mixing TMR-pS259-Raf with increasing concentra-
tions of hexaHis-14-3-3 isoforms led to progressively increased
An HTS Assay for 14-3-3 Proteins
Journal of Biomolecular Screening 11(3); 2006www.sbsonline.org 271
polarization signals (Fig. 1D). In this solution-based assay,
isoforms of 14-3-3 proteins exhibited a slightly different binding
(η), 1.43 ± 0.04 µM (σ), 1.73 ± 0.07 µM (τ), 1.89 ± 0.04 µM (β),
and 8.04 ± 0.55 µM (ε), respectively. The similarity in affinity for
phosphopeptide among different isoforms of 14-3-3 reflects the
highly conserved nature of the peptide binding site on 14-3-3, its
amphipathic groove.21Whether the detected difference between
epsilon and other isoforms is physiologically relevant remains to
teins in general, very likely including yeast and plant 14-3-3
To refine the 14-3-3 FP assay, we tested a range of different
concentrations of the fluorescent Raf probe for 14-3-3 binding
mately 150 mP and a Kd of approximately 0.4 to 0.5 µM (0.53 ±
oftheprobepeptidewasused (Fig.2B).Importantly,an S:Nratio
of greater than 9 and a Z′ factor of greater than 0.5 were achieved
under these conditions, demonstrating a robust assay even with a
fluorophore concentration as low as 0.5 nM or as high as 50 nM
14-3-3 binding. High probe concentration may be required under
interference of the assay components such as the added com-
pounds in HTS.
Examination of parameters that may
influence the robustness of the assay
14-3-3 affinity measurements and particularly for HTS. Thus, we
ing. TMR-pS259-Raf-peptide and GST-14-3-3γ were mixed and
riod (Fig. 3). The results show that the assay is indeed very stable
as revealed by virtually unchanged mP values and assay dynamic
a large number of assay plates for scheduled reading of HTS
DMSO is a common solvent used in dissolving many natural
and organic compounds as employed in small-molecule libraries.
Therefore, the 14-3-3 FP assay must be able to tolerate the pres-
ence of DMSO if it is to be used for screening 14-3-3 modulators
with a compound library. Increasing amounts of DMSO were
added to the assay. Itappears thatup to 10% DMSO did notshow
any significant changes in mP signal, Kd, or dynamic range (Fig.
4A). Further increase of the DMSO concentration slightly in-
Du et al.
272www.sbsonline.org Journal of Biomolecular Screening 11(3); 2006
cence polarization (FP) assay. (A) 5/6 carboxytetramethylrhodamine
ous concentrations of GST-14-3-3γ or GST proteins in 384-well black
microplates with a total volume of 50 µL. The FP signals were recorded
triplicate. (B) The assay window was defined by subtracting free peptide
model FP = ([14-3-3] • Bmax)/([14-3-3] + Kd) using nonlinear regression
analysis, where Bmaxis the maximal binding (Prism 4.0; Graphpad). (C)
The signal-to-noise (S:N) ratios and the Z′ factors of the assay were ob-
various concentrations of hexaHis-tagged 14-3-3 isoforms as indicated.
Experiments were performed and analyzed as described in panel B.
Validation of the 14-3-3 FP assay
with known peptide antagonists
To further validate the FP assay format for 14-3-3 studies and
ing several known peptide antagonists of 14-3-3. To achieve the
desired sensitivity, the concentrations of fluorescent Raf peptide
probe and 14-3-3 protein were carefully chosen to maximize the
difference between the highest and lowest polarization values. A
concentration of fluorescent Raf peptide probe of 1 nM was se-
tensity, which is expected to overcome any potential interference
of weakly fluorescent compounds. A well-characterized 14-3-3
R18 isa20–amino acid, unphosphorylated peptideobtained from
phagedisplay librariesthatexhibitsahigh affinity for 14-3-3 pro-
teinsin asolid-phaseassay (Kd of70-90 nM).15Asshown in Fig-
ure5,R18 competitively decreased theFPsignalwhen incubated
1.4 ± 0.2, 2.4 ± 0.4, 3.5 ± 0.6, and greater than 10 µM were ob-
ues of the peptide competitors were also increased (Fig. 5B).
sensitivity to inhibitors. To test the specificity of R18, a mutated
form of R18, R18Lys, was used. In R18Lys, 2 negatively charged
An HTS Assay for 14-3-3 Proteins
Journal of Biomolecular Screening 11(3); 2006 www.sbsonline.org 273
ent concentrations of TMR-pS259-Raf peptide, and the millipolarization
(mP) was measured and plotted as described in the legend to Figure 1.
fluorescence polarization (FP) assay.
Effect of probe peptide concentration on assay performance.
(FP) assay. FP experiments were performed as described in the legend to
(1-42 h) before the FP signals were recorded. (A) Recorded
several time intervals was plotted. (B) Assay windows of the FP reaction
with increasing concentrations of GST-14-3-3γ.
Temporal stability of the 14-3-3 fluorescence polarization
with 0.5 µM of 14-3-3γ), and the FP signal remained unchanged
conditions for competitive inhibitor studies, as required for HTS.
Two additional 14-3-3 peptide antagonists were tested in this
3-3 client proteins, ASK1 and Raf-1. Biotin-pS259-Raf is a
nonfluorescent version of the probe peptide. The pS967-ASK1
been shown to inhibit 14-3-3/client-protein interactions. Indeed,
biotin-pS259-Raf and 1.8 ± 0.2 µM for pS967-ASK1 (Fig. 5C).
Du et al.
274www.sbsonline.org Journal of Biomolecular Screening 11(3); 2006
3-3γ was performed in the presence of DMSO (0%, 1%, 2%, 4%, and
temperature (RT) for 1 h.
tive control. FP signals derived from the interaction of TMR-pS259-Raf
centrations of R18 or its mutant derivative. Percentage FP signal in the
R18 samples was expressed relative to the 14-3-3/Raf probe FP signal in
ing of TMR-pS259-Raf peptide to increasing concentrations of GST-14-
3-3γ. (C) Assay validation with 14-3-3 recognition peptides from ASK1
and Raf-1. Nonfluorescent biotin-pS259-Raf and pS967-ASK1 were in-
cubated with GST14-3-3γ and the peptide probe, TMR-pS259-Raf-1, as
Ability to measure competitive inhibition of 14-3-3/Raf pep-
FP assay, which is particularly suited for HTS of 14-3-3 modula-
tors. The tested 14-3-3 peptide inhibitors, such as R18, are valu-
able as positive controls in HTS applications.
High-throughput format development
of the assay in 384-well black microplates in a total volume of 50
to-plate variations were determined from experiments conducted
on differentdays.In our test,eachmicroplatecontained an identi-
cal set of control reactions with 8 samples each of the following:
1) free TMR-pS259-Raf peptide (1 nM) alone gives minimal mP
window, 3) TMR-pRaf with 14-3-3 in the presence of R18 (10
µM) demonstrates the minimal mP seen with maximal inhibition,
tial positives in the primary screenings. Assay performance from
forty 384-well microplates based on data from control samples 1)
and 2) above is summarized online (see Supplemental Figures 1
and 2; http://jbx.sagepub.com/cgi/content/full/11/3/269/DC1).
Both the free and bound peptide samples remained stable with
minimal fluctuation. The average FP signal of free TMR-pS259-
Raf peptide controls was 62 mP with a standard deviation of 1.8,
and the average for bound peptide controls was 143 mP with a
from each plate, were consistently higher than 10. The Z′ factor
ranged from 0.7 to 0.9, demonstrating a robust and consistent as-
ity for HTS.
To test whether the 14-3-3 FP assay could be miniaturized for
potential use of the 1536-well plate format, we examined its per-
assay component were maintained in a volume of 20 µL. As de-
recorded with the Analyst plate reader in a 384-well plate. An as-
suggesting a robust assay suitable for HTS. Further evaluation of
gest the feasibility of using the 14-3-3 FP assay in an ultra-HTS
has been developed and validated for studying 14-3-3 proteins.
The interaction of 14-3-3 with a fluorescently tagged recognition
motif from Raf-1, TMR-pS259-Raf, was used as a model system.
Binding of 14-3-3 proteins to the Raf peptide probe gave rise to
large polarization signals with robust performance. GST-tagged
and hexaHis-tagged 14-3-3 proteins exhibited similar affinity in
the need for proteolytic cleavage. This is a general 14-3-3 assay
any 14-3-3 binding peptide from its client proteins. Experiments
3-3 interaction and also show that the assay can be used to search
for compounds that disrupt 14-3-3/ligand binding. Because of its
excellent stability, DMSO tolerance, and simplicity, this assay is
particularly suited for HTS. In this format, it has an excellent S:N
the assay was miniaturized to a volume of 20 µL with excellent
plate format for ultra-HTS of small-molecule 14-3-3 inhibitors.
Theavailability of thiswell-designed, 1-step FP assay isexpected
Li for helpful discussions, and members of the Fu laboratory for
constructive suggestions. This work was supported in part by
grants from the National Institutes of Health/National Institute of
General Medical Sciences (R01 GM53165 to H.F.) and a Lung
Cancer program seed grant from Winship Cancer Institute.
Yuhong Du is a recipient of the Emory Drug Development and
Pharmacogenomics Academy Research Fellowship.
regulation. Annu Rev Pharmacol Toxicol 2000;40:617-647.
Yaffe MB: How do 14-3-3 proteins work? Gatekeeper phosphorylation and
the molecular anvil hypothesis. FEBS Lett 2002;513:53-57.
molecular interference. Cell Signal 2000;12:703-709.
Dougherty MK, Morrison DK: Unlocking the code of 14-3-3. J Cell Sci
ity of 14-3-3 isoform dimer interactions and phosphorylation. Biochem Soc
Muslin AJ, Tanner JW, Allen PM, Shaw AS: Interaction of 14-3-3 with sig-
naling proteins is mediated by the recognition of phosphoserine. Cell
An HTS Assay for 14-3-3 Proteins
Journal of Biomolecular Screening 11(3); 2006www.sbsonline.org275
7. Yaffe MB, Rittinger K, Volinia S, Caron PR, Aitken A, Leffers H, et al: The Download full-text
structural basis for 14-3-3:phosphopeptide binding specificity. Cell
Pozuelo Rubio M, Geraghty KM, Wong BH, Wood NT, Campbell DG,
trafficking. Biochem J 2004;379(Pt 2):395-408.
Meek SE, Lane WS, Piwnica-Worms H: Comprehensive proteomic analysis
of interphase and mitotic 14-3-3-binding proteins. J Biol Chem
functional, and domain-based analysis of in vivo 14-3-3 binding proteins in-
volved in cytoskeletal regulation and cellular organization. Curr Biol
Mol Cell Cardiol 2004;37:633-642.
Hermeking H: The 14-3-3 cancer connection. Nat Rev Cancer 2003;3:931-
MH, et al: Interaction of Akt-phosphorylated ataxin-1 with 14-3-3 mediates
neurodegeneration in spinocerebellar ataxia type 1. Cell 2003;113:457-468.
Wang B, Yang H, Liu Y, Jelinek T, Zhang L, Ruoslahti E, et al: Isolation of
high-affinity peptide antagonists of 14-3-3 proteins by phage display. Bio-
J Biol Chem 2001;276:45193-45200.
binds a phosphorylated Raf peptide and an unphosphorylated peptide via its
conserved amphipathic groove. J Biol Chem 1998;273:16305-16310.
cardiac fructose-2,6-bisphosphate kinase/phosphatase. Embo J
Arkin MR, Wells JA: Small-molecule inhibitors of protein-protein interac-
Fotouhi N, Graves B: Small molecule inhibitors of p53/MDM2 interaction.
Curr Top Med Chem 2005;5:159-165.
molecule Smac mimic potentiates TRAIL- and TNFalpha-mediated cell
death. Science 2004;305:1471-1474.
interact with 14-3-3zeta through a common site involving lysine 49. J Biol
Banks P, Gosselin M, Prystay L: Fluorescence polarization assays for high
throughput screening of G protein-coupled receptors. J Biomol Screen
polarization based Src-SH2 binding assay. Anal Biochem 1997;247:77-82.
Owicki JC: Fluorescence polarization and anisotropy in high throughput
screening: perspectives and primer. J Biomol Screen 2000;5:297-306.
screening assays using fluorescence polarization: nuclear receptor-ligand-
binding and kinase/phosphatase assays. J Biomol Screen 2000;5:77-88.
fluorescence polarization. Anal Biochem 1997;249:29-36.
polarization assay of protein kinase C. J Biomol Screen 2000;5:23-30.
Jameson DM, Mocz G: Fluorescence polarization/anisotropy approaches to
Mol Biol 2005;305:301-322.
ZhangL,ChenJ,FuH: Suppressionofapoptosis signal-regulatingkinase 1-
Current Protocols in Molecular Biology. New York: John Wiley, 1987.
Fu H, Coburn J, Collier RJ: The eukaryotic host factor that activates
family. Proc Natl Acad Sci USA 1993;90:2320-2324.
Zhang JH, Chung TD, Oldenburg KR: A simple statistical parameter for use
in evaluation and validation of high throughput screening assays. J Biomol
ligand interaction. Proteins 2002;49:321-325.
Ferl RJ: 14-3-3 proteins: regulation of signal-induced events. Physiol Plant
Address reprint requests to:
Haian Fu, Ph.D.
Department of Pharmacology
Emory University School of Medicine
and Emory Chemistry-Biology Discovery Center
Atlanta, GA 30322
Du et al.
276 www.sbsonline.orgJournal of Biomolecular Screening 11(3); 2006