M lysis buffer

H1975 cells were incubated with HAD-B1 and with afatinib in RPMI1640 (2% fetal bovine serum, 1× antibiotics) for 72 hours, after which the cells were harvested to extract the proteins. The cell lysates were prepared in Lysis-M buffer (Roche) containing protease and phosphatase inhibitor cocktails (both from Roche). The cell lysates were clarified by centrifugation. Lysates containing 30 µg of protein were loaded into each well and separated through 12% sodium dodecyl sulfate–gel electrophoresis. Gels were soaked in transfer buffer (16 mM Tris-HCl, 30-mM glycine, and 20% methanol), and proteins were then transferred to polyvinylidene difluoride membranes. Nonspecific binding sites were blocked by incubation with 5% nonfat dry milk in PBST (137 mM NaCl, 27 mM KCl, 100 mM Na₂HPO₄, 20 mM KH₂PO₄, 0.05% Tween20, and pH 7.4). The polyvinylidene difluoride membranes were then incubated with primary antibodies against p-ERK1/2 (1:1000), ERK1/2 (1:1000), p-EGFR (1:1000), EGFR (1:1000), p16 (1:1000), and β-actin (1:10000) in PBST containing 5% bovine serum albumin and 5% nonfat dry milk at 4°C overnight. Membranes were washed with PBST and incubated with secondary antibodies (anti-mouse at 1:16 000, anti-rabbit at 1:16 000). Signals were then developed by using an enhanced chemiluminescence Western blotting detection kit and were exposed to X-ray films.