P p70s6k thr421 ser424

The cells were lysed by boiling in SDS‐loading buffer containing 0.1 M Tris (pH 6.8), 16% v/v glycerol, 3.2% w/v SDS, 10% v/v β‐mercaptoethanol and 0.005% w/v bromophenol blue. Sample loading was normalized according to protein concentration determined by DC protein assay (Bio‐Rad). Cell lysates were subjected to SDS‐PAGE and electroblotted to PVDF membrane. The blots were probed with the pAkt (Ser473)‐, Akt‐, p‐p70S6K (Thr421/Ser424)‐ and pGSK3beta (Ser9)‐specific antibodies (Cell Signaling Technology, Beverly, MA, USA) or the anti‐alpha‐tubulin antibody (Sigma‐Aldrich) according to instructions of manufacturers. The blots were developed using secondary antibodies conjugated to peroxidase (Cell Signaling Technology; Sigma‐Aldrich) and standard ECL using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA).

Equivalent total protein lysates were loaded in each lane, run on SDS-PAGE, transferred to PVDF membranes, blocked with 5% skim milk, incubated with primary antibodies overnight at 4°C, including anti-p-EGFR Y1092(#ab40815, Abcam, Cambridge, UK), EGFR (#ab52894, Abcam, Cambridge, UK), β-catenin (#ab32572, Abcam, Cambridge, UK), cyclin B1 (#4135, Cell Signaling Technology Inc., MA, USA), p-p70 S6K Thr421/ Ser424 (#9204, Cell Signaling Technology Inc., MA, USA), p70 S6K (#2708, Cell Signaling Technology Inc., MA, USA), p-Erk1/2 Thr202/ Tyr204 (#9101, Cell Signaling Technology Inc., MA, USA), Erk1/2 (#4696, Cell Signaling Technology Inc., MA, USA), p-STAT3 Tyr705 (#9145, Cell Signaling Technology Inc., MA, USA), STAT3 (#569388, Calbiochem, CA, USA), p-STAT1 Y701 (#ab29045, Cell Signaling Technology Inc., MA, USA), STAT1 (#06–501, EMD Millipore Corporation, CA, USA), GAPDH (#ab181602, Abcam, Cambridge, UK), and β-actin (#A5441, Sigma-Aldrich, MO, USA), and observed under a chemiluminescence system.

Cell protein extraction, solubilization, and analysis by 1-D PAGE were performed as previously described [41 (link)]. Antibodies against HER2; p-HER2 ^Tyr1221/1222; p70S6K; p-p70S6K ^{Thr421/Ser424}; Akt; p-Akt ^Ser473; p44/42 MAPK; p-p44/42 MAPK; caspase-7 and 9; cyclin A and B1; Rb; p-Rb were from Cell Signaling Technology (Beverly, MA). Antibody against cytochrome-c (7H8) was form Santa Cruz Biotechnology Inc. (Dallas, TX). Antibodies against actin was from Sigma–Aldrich (St Louis, MO). Antibody against GAPDH was from Ambion (Austin, TX). HRP-conjugated secondary antibodies were from Pierce (Rockford, IL) and chemiluminescence system (ImmobilionTM Western Cemiluminescent HRP Substrate), was from Millipore (Temecula, CA).