Phospho bim s69

S116836 (chemical structure, Fig. 1A) was rationale designed and synthesized in our lab [26 ]. S116836 was dissolved in DMSO at a stock concentration of 20 mM and stored in aliquots at −20°C. U0126 and LY294002 were purchased from Calbiochem (San Diego, CA). Cycloheximide (CHX) was bought from Sigma-Aldrich (St. Louis, MO). MG132 was obtained from EMD Bioscience (Billerica, MA). Antibodies against poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP), pro-caspase3, X-linked inhibitor of apoptosis protein (XIAP) and cytochrome c were from BD Biosciences (San Jose, CA). Antibodies against phospho-PDGFRα (Y1018), phospho-Erk1/2 (T202/Y204), Erk1/2, phospho-AKT (S473), AKT, caspase-8, caspase-9, Bax and phospho-Bim (S69) were from Cell Signaling Technology (Beverly, MA). Antibodies against phospho-STAT3 (Y705), STAT3 and PDGFRα were from EMD Millipore Upstate (Billerica, MA). Anti-Mcl-1 (S-19) was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Bim was from Stressgen Enzo Life Sciences (Plymouth Meeting, Pennsylvania). Anti-Survivin was from Novus Biologicals (Littleton, CO, USA). Anti-active-caspase-3 and Anti-β-actin were from Sigma-Aldrich (St. Louis, MO). Anti-mouse immunoglobulin G and anti-rabbit immunoglobulin G horseradish peroxidase-conjugated antibodies were purchased from Pierce Biotechnology (Los Angeles, CA, USA).

Specified antibodies to the following proteins were used for immunoblotting (IB), immunoprecipitation (IP), immunofluorescence (IF), and immunohistochemistry (IHC): phospho-Akt1(S473) (1:2000, IB; ab81283, abcam), phospho-Bad(S112) (1:1000, IB, no. 5284, Cell Signaling), phospho-BIM(S69) (1:400, IB; no. 4581, Cell Signaling), phospho-Elk1 (S383) (1:1000, IB; no. 9181, Cell Signaling), phospho-Elk1 (S383) (1:1000, IB; ab34270, abcam), phospho-ERK1/2(TEY) (1:1000, IB; no. 9101L, Cell Signaling), ERK1/2 (1:1000, IB; no. 9102, Cell Signaling), phospho-ERK1/2(T188) (1:1000, IB; 1:500 IHC; Lorenz et al., 2009 (ref. ^{13 (link)}) or A010-40AP, Badrilla), Flag (1:10,000, IB or 1:200, IF; IP 5.6μg/sample;; F3165, Sigma), phospho-Foxo3a(S294) (1:1000, IB; no. 5538, Cell Signaling), Gβ (1:5000, IB; sc-378, Santa Cruz Biotechnology), HA (1:5000, IB; IP 5μg/sample; MMS-101R, Covance), HA (1:500, IF; no. 3724, Cell Signaling), Max (1:200, IF; sc-197, Santa Cruz Biotechnology), c-Myc (1:1000, IB; sc-789, Santa Cruz Biotechnology), c-Myc (1:350, IF; M4439, Sigma), phospho-p70(S6) (1:1000, IB; no. 9204, Cell Signaling), and phospho-p90RSK(S380) (1:5000, IB; ab32203, abcam).