Anti py

Cells were lysed at 4℃ in lysis buffer (50 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1% Nonidet P-40, and protease inhibitor cocktail). Following a 40-min incubation on ice, protein extracts were centrifuged at 15,000 × g for 10 min at 4℃. The amount of protein in the clarified supernatants was measured using Bradford Reagent (Sigma). Lysates were diluted with sodium dodecyl sulfate (SDS) sample buffer (0.4 M SDS, 0.4 M Tris-HCl, 40 mM EDTA, 50% glycerol, and bromophenol blue). The lysates were separated using SDS polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane using an iBLOT system (Invitrogen). Membranes were blocked in Tris-buffered saline containing 0.1% Tween 20 and 5% nonfat dry milk, and then incubated with the following specific primary antibodies: anti-EGFR, anti-IGF-1R, anti-pEGFR (Tyr1045), anti-p-IGF-1R (Tyr1280), anti-pY (Santa Cruz). Horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG was used as the secondary antibody (Dako). Immunoreactive protein was visualized by chemiluminescence using the SuperSignal^® West Pico Luminol/Enhancer Solution (Thermo Scientific). Equal protein loading was evaluated using an anti-β-actin antibody (Sigma).