Cell lysate preparation, Western blotting, and densitomeric analysis

Adherent cells were lysed in cell lysis buffer, and Western blot analysis was performed as previously described (8). Cells were treated with the proteasome inhibitor MG132 (N-carbobenzyloxy-L-leucyl-L-leucyl-L-leucinal) (5 μM) for 16 hours before the lysis of cells for the analysis of SOCS4 and SOCS5 proteins. Antibodies used for Western blotting were as follows: anti-pSTAT3 (Y705), total STAT3, pERK (p44/42, T202/Y204), pAKT (S473), total AKT, pS6 (S240/244), S6, pEGFR (Y1068), total EGFR, L858R-EGFR– or del19-EGFR–specific antibodies, anti-JAK2 (Cell Signaling Technology), and anti-JAK1 (BD Biosciences); anti–total ERK, EGFR, Myc, ubiquitin and horseradish peroxidase (HRP)–conjugated anti-rabbit IgG, HRP-conjugated anti-mouse IgG, HRP-conjugated anti-rabbit IgG, SOCS4, and SOCS5 (Santa Cruz Biotechnology); anti–c-MET (Invitrogen); and anti–α-tubulin (Sigma-Aldrich). Densitometric quantification of high-resolution immunoblot images was performed using an analysis program, ImageJ (developed by W. Rasband, Research Services Branch of the National Institute of Mental Health). Analyses of the phosphorylation status of RTK and ErbB family members were performed using the RayBio Human RTK and EGFR Phosphorylation Array kits according to the manufacturer’s instruction (RayBiotech).