Antibody specific for p21

Amlodipine and gefitinib were purchased from Selleck Chemicals (Houston, TX, United States) and TopScience (Shanghai, China), respectively. Propidium iodide (PI) was obtained from Sigma-Aldrich (St. Louis, MO, United States). The Annexin V/FITC-PI apoptosis detection kit was obtained from BD Biosciences (San Jose, CA, United States of America). RPMI 1640 and FBS were purchased from Biological Industries (Beit Haemek, Israel). The enhanced chemiluminescence (ECL) reagent was purchased from Thermo Fisher Scientific (Waltham, MA, United States). Antibodies against phospho-EGFR, phospho-PDK1 (Ser241), PDK1, phospho-Akt (Ser473), phospho-Akt (Thr308), Akt, phospho-mammalian target of rapamycin complex 1 (mTORC1) (Ser2448), mTOR, phospho-glycogen synthase kinase 3 (GSK-3) β, phospho-p70 S6K (Thr389), p27, cyclin D1, phospho-retinoblastoma protein (Rb), phospho-extracellular signal-regulated kinase (ERK) 1/2, phospho-c-Raf, β-actin, and horseradish peroxidase (HRP)-conjugated goat anti-rabbit and horse anti-mouse secondary antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA, United States). Antibody specific for p21 was obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, United States).

Total cell protein was dissolved in RIPA buffer containing 150 mM NaCl, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0 (Sigma-Aldrich Corp) with 1× proteinase inhibitor cocktail (Sigma-Aldrich Corp) and phosphatase inhibitor cocktail (Pierce). The lysate was incubated on ice for 15 minutes and centrifuged at 12,000 g for 10 minutes. The amount of protein in the supernatant was quantified using the BCA Protein Assay kit (Pierce). Fifty μg of total protein was separated on a 10% SDS-PAGE gel and then transferred to PVDF membranes (Bio-Rad Laboratories) by electroblotting. Membranes were incubated at 4 °C overnight with a 1:1,000 dilution of antibody specific for γH2AX (Novus Biologicals), a 1:500 dilution of antibody specific for P21 (Santa Cruz Biotechnology, Inc), and a 1:1,000 dilution of antibody specific for β-actin antibody (Santa Cruz Biotechnology, Inc).