Anti total egfr

Cells were washed twice with phosphate buffered saline (PBS) before being lysed on ice for 40 minutes with lysis buffer containing 50 mmol/L HEPES buffer, 150 mmol/L NaCl, 1 mmol/L EDTA (pH 8.0), 1 mmol/L EGTA (pH 8.0), 1% IGEPAL CA-630, 0.5% Triton X-100, 10 mmol/L NaF, 2 mmol/L Na₃VO₄, 10 mmol/L β-glycerophosphate, and 1% protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). The lysate was centrifuged at 16,000 × g at 4°C for 10 minutes. Ten to fifty micrograms of total protein for each sample were separated by 8%~12% SDS-PAGE and transferred onto an Immobilon membrane (Millipore Inc, Billerica, MA.), and the desired proteins were probed with corresponding antibodies. Mouse anti-HPV16 E7 (1:1000 dilution) and anti-total EGFR (1:1000) were purchased from Santa Cruz, mouse anti-human actin (1:10000 dilution) from Sigma, anti-β catenin antibody from BD Pharmingen (San Jose, CA), and anti-phospho EGFR (tyrosine 1173) antibody from Cell Signaling (Danvers, MA). Horseradish peroxidase– conjugated secondary anti-mouse and anti-rabbit IgG (H+L) were obtained from Promega. Bound antibody was detected using the SuperSignal West Pico Chemoluminescence system (Pierce, Inc. Rockford, IL).