2.3. Western Blot Analysis

Whole-cell protein extracts were prepared using lysis buffer (1% Triton, 10% glycerol, 50 mM Tris, 2 mM EDTA, and 150 mM NaCl) supplemented with fresh protease inhibitors ({"type":"entrez-protein","attrs":{"text":"PIA32961","term_id":"1273151213","term_text":"PIA32961"}}PIA32961, Thermo Fisher Scientific) and incubated 1 h at room temperature, followed by centrifugation. Proteins were dosed using a Bradford assay (Bio-Rad, Hercules, CA, USA). A total of 30 μg of whole-cell lysate was loaded on 6% sodium dodecyl-sulphate-polyacrylamide gel electrophoresis (SDS-PAGE, Bio-Rad) and transferred to nitrocellulose membrane using the Trans-Blot Turbo Transfer System (Bio-Rad). The membrane was immunoblotted with either rabbit monoclonal anti-EGFR (1:10,000, EP38Y, ab52894, Abcam Inc. Cambridge, UK), mouse monoclonal anti-ERBB2 (1:750, 3B5, OP15L, Calbiochem, San Diego, CA, USA), rabbit monoclonal anti-ERBB3 (1:200, D22C5, #12708, Cell Signaling Technology, Danvers, MA, USA), or mouse monoclonal anti-ERBB4 (1:500, C-7, sc8050, Santa Cruz Biotechnology, Dallas, TX, USA) antibodies. Each primary antibody was diluted in Tris-buffered saline tween 20 (TBS-T) containing 5% fat-free milk powder. α-tubulin was used as a loading control (DM1A, sc-32293, Santa Cruz Biotechnology). Immunoreactive bands were detected by enhanced chemiluminescence (ECL, GE Healthcare, Little Chalfont, UK).