Ecl western blotting detection system

The AGS cells were serum-starved overnight in DMEM with 0.2% FBS and treated as required (see above). The plates were then washed with ice-cold Ca^2+/Mg²⁺-free phosphate-buffered saline and lysed in freshly prepared lysis buffer (2 mM Na₃VO₄, 4 mM sodium pyrophosphate, 10 mM sodium fluoride, 50 mM HEPES pH 7.9, 100 mM NaCl, 10 mM EDTA, 1% Triton X-100, 2 µg/mL leupeptin, 2 µg/mL aprotinin, 1 mM phenylmethylsulfonyl fluoride). Protein concentrations were determined using the BCA protein assay (Thermo Fisher Scientific). The cell extracts were subjected to 4% to 20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (pre-cast gels; Bio-Rad Laboratories, Hercules, CA, USA), with the separated proteins transferred to polyvinylidene difluoride membranes. After blocking the nonspecific binding sites with albumin or nonfat dry milk, the membranes were incubated with antibodies against the following proteins (as required): phospho-p44/42 MAPK, phospho-Akt, and phospho-EGFR (Tyr1068) (Cell Signaling Technology); EGFR (Santa Cruz Biotechnology, Santa Cruz, CA, USA); β-actin (Sigma-Aldrich) (protein loading control). Secondary antibodies were HRP-conjugated anti-rabbit or anti-mouse (Bethyl Laboratories, Montgomery, TX, USA), and the immune complexes were visualized using the ECL Western blotting detection system (Euroclone).