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Supplementary Figure Legends
Figure S1.
Loss of BimEL affords only transient protection against serum
withdrawal in iBMK cells. WT and Bim-/- iBMK cells were serum-starved (SF) or
treated with 300nM Paclitaxel in complete media (Pac) for up to 48 hours. At the times
indicated attached and floating cells were collected and assayed for viability by Trypan
blue exclusion. Results are expressed as the percentage viability against untreated cells
(t=0).
Figure S2. Rapid and ERK1/2-dependent dissociation of BimEL from Mcl-1 and
Bcl-xL in iMEFs and CR1-11 cells. (A) CCl39 cells were maintained in complete
medium with 10% FBS (CM) or serum starved for 6 hours before re-stimulating with
fresh 10% FBS or 10nM thrombin for 15 minutes. Whole cell extracts were then
prepared, normalised for protein content and boiled directly (Input WCE) or used for
immunoprecipitation of Mcl-1. Input and IP samples were then immunoblotted with
antibodies to Mcl-1, Bim, P-ERK1/2 and ERK1/2. (B) WT iMEFs in complete medium
(CM) were serum starved in the presence of 20µM U0126 (SF/U0) and then washed
thoroughly before stimulating with FBS for 1 hour in the presence of 10µM MG132.
Whole cell extracts were then prepared, normalised for protein content and boiled
directly (Input WCE) or used for immunoprecipitation of Bcl-xL. Input and IP samples
were then immunoblotted with antibodies to Bcl-xL, Bim, P-ERK1/2 and ERK1/2. Total
and Bcl-xL-associated BimEL were quantified from live ECL images. Results are taken
from a single representative experiment. (C) Cycling CR1-11 cells (CCl39 cells
expressing ∆Raf-1:ER*) in complete medium (CM) were serum starved (SF) for 6 hours
to induce Bim expression and formation of BimEL/Bcl-xL complexes. These cells were
then stimulated for 1 hour with 100nM 4-hydroxytamoxifen (4-HT) ± 20µM U0126.
Whole cell extracts were then prepared, normalised for protein content and boiled
directly (Input WCE) or used for immunoprecipitation of Bcl-xL. Input and IP samples
were then immunoblotted with antibodies to Bcl-xL, Bim, P-ERK1/2 and ERK1/2.
Results are taken from a single experiment representative of three giving identical results.
The asterisk * in indicates cross-reactivity with the antibody light chain used in the IP.
Figure S3.
existing
FBS accelerates BimEL turnover but BimEL dissociation from pre-
BimEL/Mcl-1
complexes
does
not
require
proteasome-dependent
degradation of BimEL. (A & B) CCl39 cells were serum starved overnight, treated with
emetine for 30 minutes and then left serum free or stimulated with FBS for the times
indicated. (A) Whole cell lysates were probed by western blot for Bim, Mcl-1, Bcl-xL,
P-ERK1/2 and ERK1/2. Of the three Bcl-2 family proteins, only BimEL expression levels
were accelerated by the re-stimulation with FBS and this was quantified in (B). (C)
CCl39 cells were serum starved in the presence of 20µM U0126 (SF/U0) and then
washed thoroughly before stimulating with FBS for 1 hour in the presence of 10µM
MG132 with or without cycloheximide. Whole cell extracts were then prepared,
normalised for protein content and boiled directly (Input WCE) or used for
immunoprecipitation of Mcl-1. Input and IP samples were then immunoblotted with
antibodies to Mcl-1, Bim, P-ERK1/2 and ERK1/2. The asterisk * in indicates crossreactivity with the antibody light chain used in the IP.
Figure S4. ERK1/2-dependent dissociation of BimEL from Mcl-1 is reversible when
ERK1/2 is inhibited. CR1-11 cells (CCl39 cells expressing ∆Raf-1:ER*) in complete
medium (CM) were serum starved (SF) for 6 hours to induce Bim expression and
formation of BimEL/Mcl-1 complexes. These cells were then stimulated with 100nM 4HT for 2 or 4 hours or for 2 hours followed removal of 4-HT and addition of serum free
medium containing 20µM U0126 to inactivate ERK1/2. (A) Whole cell extracts were
prepared, normalised for protein content and boiled directly (Input WCE) or used for
immunoprecipitation of Mcl-1. Input and IP samples were then immunoblotted with
antibodies to Mcl-1, Bim, P-ERK1/2 and ERK1/2. The asterisk * in indicates crossreactivity with the antibody light chain used in the IP. (B) Total and Mcl-1-associated
BimEL were quantified from live ECL images. (C) Cells treated in parallel to (A) were
separated into the cytosolic and heavy membrane fractions, separated by SDS-PAGE and
Western blotted with antibodies to Mcl-1, Bim, P-ERK1/2 and ERK1/2, COX-IV and
tubulin. Results are taken from a single representative experiment.
Figure S5. Activation of ∆Raf-1:ER* fails to promote dissociation of Puma from
Mcl-1. HR1 cells (HEK293 cells expressing ∆Raf-1:ER*) were transfected with FlagPuma in complete medium (C). After 24 hours cells were switched to fresh complete
medium (C) or serum free (SF) medium for 6 hours before stimulating with 4-HT form
15 minutes. U = Untransfected control. Whole cell extracts were normalised for protein
content and either boiled directly (Input WCE) or used for immunoprecipitation of Mcl-1.
Samples fractionated by SDS-PAGE were immunoblotted with antibodies to Mcl-1,
Flag(Puma), P-ERK1/2 and ERK1/2. Results are taken from a single experiment
representative of three giving identical results.