Anti bad

Cells were washed with ice-cold Phosphate-Buffered Saline (PBS) and then lysed in Triton X-100-containing lysis buffer. The composition of the lysis buffer was as follows: 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 2.5 mM EDTA, 2.5 mM EGTA, 20 mM NaF, 1 mM Na₃VO₄, 20 mM Sodium β-Glycerophosphate, 10 mM Sodium Pyrophosphate, 0.5% Triton X-100, Roche protease inhibitor cocktail and 0.1% β-Mercaptoethanol. Lysates were precleared by centrifugation before use for Western blotting. Equal amounts of protein were loaded for Western blot analysis. All the following antibodies used were obtained from Cell Signaling Technology: anti-phospho-p38 (Thr180/Tyr182), anti-p38, anti-phospho-SAPK/JNK (Thr183/Tyr185), anti-SAPK/JNK, anti-phospho-ERK1/2 (Thr202/Tyr204), anti-ERK1/2, anti-Mcl-1, anti-phospho-Bad (Ser112), anti-Bad, anti-phospho-MEK1/2, anti-MEK1/2, anti-Grb2, anti-phospho-Akt (Thr308), anti-Akt, anti-phospho-STAT3 (Tyr705), anti-STAT3, anti-calnexin, anti-phospho-p70 S6K1 (Thr389), anti-p70S6K1, except for anti-p27 (BD Biosciences), anti-HIF-1α (BD Transduction Laoratories), anti-GAPDH (US Biological), anti-Glut1 (Abcam), anti-α-tubulin (Molecular Probes), anti-β-actin (Sigma), anti-FLAG M2 (Sigma), anti-V5 (Serotec) and anti-HA (Roche).

Cells were lysed in freshly prepared RIPA buffer supplemented with 1x Halt Protease/Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). Cleared lysates were resolved by SDS-PAGE, transferred to PVDF membrane and incubated with primary antibodies. The antibodies applied in the current study are as follows: anti-BCL-2 (M0887, DAKO), anti-BID (#2002, Cell Signaling Technology), anti-BAG3 (NBP2-27398, NOVUS), anti-BOK (AF6067, R&D System), anti-BAK (SC-7873, Santa Cruz), anti-BAD (610391, BD Bioscience), anti-CASP9 (#9502, Cell Signaling Technology), anti-CYCS (556433, BD Bioscience), anti-BIRC5 (SURVIVIN) (#2802, Cell Signaling Technology), anti-XIAP (#2042, Cell Signaling Technology), GAPDH (AF5718, R&D System), anti-SNAI2 (SLUG) (ab27568, Abcam), anti-SOX9 (ab26414, Abcam), anti-total CD44 (ab51037, Abcam), anti-integrin α2β3 (ab662, Abcam), anti-HIF1A (AF1935, R&D System), anti-HIF2A (AF2886, R&D System) and α-tubulin (T5168, Sigma).