Pher2 tyr1221 1222

Following treatment, cancer cells were lysed with RIPA buffer (0.1% sodium dodecylsulfate (SDS), 0.5% deoxycholate, 1% Nonidet, 100 mmol/L NaCl, 10 mmol/L Tris–HCl (pH 7.4), 0.5 mmol/L dithiotritol, and 0.5% phenylmethyl sulfonyl fluoride, protease inhibitor cocktail (HoffmannLa Roche, Basilea, Svizzera) and isolated by centrifugation at 14,000 rpm for 10 min at 4 °C. After Bradford assay quantification (Bio-Rad Hercules, CA, USA), protein lysates were subjected to SDS-PAGE and Western blot analysis.
Primary antibodies for western blot analysis against p-EGFR (Tyr1068), EGFR, p-MAPK44/42 (Thr202/Tyr204), MAPK44/42, p-AKT (Ser473), AKT, pHER2 (Tyr1221/1222), HER2, pHER3 (Tyr1289) and HER3 were obtained from Cell Signaling Technology; monoclonal anti-α-tubulin antibody (T8203) from Sigma Chemical Co. St. Louis, MO, USA. The following secondary antibodies from Bio-Rad were used: goat anti-rabbit IgG and rabbit anti-mouse IgG. Immunocomplexes were detected with the enhanced chemiluminescence kit ECL plus (Thermo Fisher Scientific, Rockford, IL, USA) using the ChemiDoc (Bio-Rad). Each experiment was done in triplicate.

Total protein was extracted from the cell lines following the standard protocol, and 20–40 μg of protein was used for immunoblotting^{27 (link)}. The blots were cut prior to hybridization with antibodies during blotting. The primary antibodies purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA) were as follows: pHER2-Tyr1221/1222 (#2249), pEGFR-Tyr1173 (#4407), EGFR (#2232), pHER3-Tyr1289 (#4791), pMET-Tyr1234/1235 (#3077), pIGF1R-Tyr1135/1136 (#3024), IGF1R (#9750), ERK (#4695), pAKT-Ser473 (#4060), pAKT-Thr308 (#4056), AKT (#9272), pH2A.X-Ser139 (#9718), cleaved PARP (#9541), and PCNA (#2586). Other primary antibodies [HER3 (sc-285), MET (sc-514148), pERK-Tyr204 (sc-7383), and PTEN (sc-6818)] were purchased from Santa Cruz Biotechnology, Inc. Anti-HER2 (ab16901) and anti-α-tubulin (T6199) antibodies were purchased from Abcam Inc. and Sigma-Aldrich, Inc., respectively. After incubation with peroxidase-conjugated secondary antibodies for 1 h at 20 °C, the protein blots were developed using an enhanced chemiluminescent reagent (Amersham Inc., Amersham, UK). Total protein visualized on the ChemiDoc™ XRS + System (Bio-Rad) and analysed on Image Lab software (Image Lab 6.0, Bio-Rad).

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).