Anti phospho erk1 2

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).