Goat anti mouse or goat anti rabbit secondary antibodies conjugated to horseradish peroxidase

Whole cell extracts were prepared by scraping cells in lysis buffer (150 mM NaCl, 5 mM EDTA, 0.5% NP40, 50 mM Tris, pH 7.5), resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific, Waltham, MA, USA). Antibodies to p-EGFR (Y1068), EGFR, p-ErbB2 (Y1221), ErbB2, p-ErbB (Y1289), ErbB3, p-IGF1R (Y1135), IGF1R, p-PDGFRβ (Y751), PDGFRβ, p-Met (Y1234/1235), Met, p-AKT (S473), pan AKT, p-ERK (T202/204), and ERK were from Cell Signaling (Danvers, MA, USA); Phospho-IRS1 (Y612) was from EMD Millipore (Burlington, MA, USA). IRS1 was from Bethyl Laboratories (Montgomery, TX, USA). PRCP was from R&D systems (Minneapolis, MN, USA). β-actin was from Santa Cruz (Dallas, TX, USA). Primary antibodies were detected with goat anti-mouse or goat anti-rabbit secondary antibodies conjugated to horseradish peroxidase (Life Technologies, Carlsbad, CA, USA), using Clarity chemiluminescence (BIO-RAD, Hercules, CA, USA). For some blots, PVDF membranes were cut into halves at the protein markers around 75–100 KD. The upper parts were immunoblotted for high molecular weight proteins, and the lower parts were immunoblotted for low molecular weight proteins.