Phospho erk1 2 thr202 tyr204

Cell lysates were prepared by washing once with cold PBS followed by lysis with a modified RIPA buffer containing 25 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM NaF, 1 % NP-40, 10 % glycerol, 2 mM Na₃VO₄, and 1X EDTA-free protease inhibitor cock-tail tablets (Roche) as previously described by Chinn et al. (2011) (link). Protein concentration was determined using the Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL, USA) according to the manufacturer’s protocol. SDS-PAGE was performed with 10–25 μg of protein loaded for each sample. Protein was transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA) and probed overnight at 4 °C with the following primary antibodies: phospho-AKT (Ser473) (#4060), AKT (#9272), phospho-ERK1/2 (Thr202/Tyr204) (#4370), ERK1/2 (#4696), EGFR (C74B9, #2646), BIM (#2819), cleaved caspase-3 (#9661; Cell Signaling Technology, Danvers, MA, USA), phospho-EGFR (Tyr1068; #44788G; Invitrogen, Carlsbad, CA, USA), PARP-1 (#sc-8007; Santa Cruz Biotechnology, Santa Cruz, CA, USA), β-actin (#A2228; Sigma-Aldrich, St. Louis, MO, USA). Blots were then incubated for 1 h at room temperature with the HRP-conjugated secondary antibodies, anti-mouse IgG (W4021) and anti-rabbit IgG (W4011; Promega, Madison, WI, USA), and visualized by chemiluminescence with Amersham ECL (GE Healthcare, Buckinghamshire, UK).