Volume 23 October 15, 2012 Pkh1/2 regulate ESCRT-I localization to MVB | 4063
centrifugations at 500 × g. The total lysate protein concentration
was quantiﬁed by Bradford reagent (Bio-Rad). Volume of total lysate
corresponding to 1 mg of proteins was brought up to 900 μl with
immunoprecipitation (IP) buffer (50 mM Tris/HCl, pH 7.5, 150 mM
NaCl, 1 mM EDTA, pH 7.5, 1 mM EGTA, pH 7.5, 1% Nonidet P-40)
supplemented with protease inhibitor (Complete Mini-EDTA Free),
and 50 μl of 50% protein G–Sepharose beads (Sigma-Aldrich, St.
Louis, MO) and 5 μl of rat anti-HA antibodies (3F10; Roche) were
added. The tubes were incubated on an overhead rotator overnight
at 4°C. The beads were washed three times with IP buffer and then
twice with 40 mM MOPS, pH 7.5.
The beads were then resuspended in 60 μl of phosphorylation
buffer (40 mM MOPS, pH 7.5, 10 mM MgCl
, 1 mM dithiothreitol).
A 10-μl amount of immune complex beads was mixed with 25 μg of
substrate protein in 10 μl of phosphorylation buffer and 2 μl of ATP
mix (1 mM ATP, 4 μCi of [γ-
P]ATP). The phosphorylation mixture
was incubated for 30 min at room temperature. The reaction was
stopped by addition of 4× Laemmli buffer containing 50 mM ATP.
The samples were boiled for 5 min and then loaded on a 10% poly-
acrylamide gel. The gel was treated for 5 min in 12.5% TCA and for
5 min in 50% (vol/vol) EtOH and 10% (vol/vol) acetic acid, dried, and
exposed on a phosphoscreen. After 24 h of exposure the screen was
scanned using a Typhoon Trio (GE Healthcare).
The equivalent of 30 OD
units of cells was lysed in 500 μl of
phosphate-buffered saline (PBS) and 0.25 M sorbitol supplemented
with protease inhibitor (Complete Mini-EDTA Free) with 500 μl of
glass beads by vortexing for 4 min at 4°C. The lysate was cleared by
two centrifugations of 3 min at 500 × g. The cleared lysate was then
spun for 10 min at 13,000 × g, generating the P13 pellet; the S13
was further spun for 1 h at 100,000 × g to generate the P100 and
S100. The P13 and P100 were resuspended in the same volume as
S100 of PBS, 0.25 M sorbitol, and 1% Triton X-100.
Equal volumes of each fraction were analyzed by SDS–PAGE,
followed by immunoblot with mouse monoclonal anti-Vps10 (Invit-
rogen, Carlsbad, CA), mouse monoclonal anti-HA (Roche), mouse
anti-ALP (Invitrogen), mouse anti-Vti1 (a gift from G. F. von Mollard,
Universität Bielefeld, Germany), and mouse anti-Pgk1 (Invitrogen)
Cells expressing the different GFP-tagged proteins were grown to
mid-exponential growth phase in selective medium before obser-
vation in the selective medium using ﬂuorescence microscopy
(Axiovert 200, 100× objective, differential interference contrast
and GFP ﬁlters [Carl Zeiss, Jena, Germany]). Images were acquired
with AxioVision (Zeiss) software using a CoolSnapHQ2 camera
(Roper Scientiﬁc, Tucson, AZ) and processed with ImageJ software
(National Institutes of Health, Bethesda, MD).
We thank Naima Belgareh-Touzé, Scott D. Emr, Rosine Haguenauer-
Tsapis, Robert C. Piper, Howard Riezman, and Gabriele Fischer von
Mollard for sharing antibodies, strains, and plasmids; J. O. De
Craene and N. Joly for critical reading of the manuscript; the Friant
laboratory’s members for support; and Romeo Ricci for support dur-
ing the revision process. This work was supported by the Centre
National de la Recherche Scientiﬁque (ATIP-CNRS 05-00932 and
ATIP-Plus 2008-3098 to S.F.), the Agence Nationale de la Recherche
(ANR-07-BLAN-0065 to S.F.), the Fondation Recherche Médicale
(FRM INE20051105238 and FRM-Comité Alsace 2006CX67-1 to
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