Western blotting analysis

MDA-MB-231 cells were washed once with cold PBS and scraped into lysis buffer (10 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na₄P₂O₇, 2 mM Na₃VO₄, 1% Triton-X 100, 10% glycerol, 0.1% sodium dodecyl sulfate (SDS), 0.5% deoxycholate, 1 mM PMSF, 5% protease inhibitor cocktail) for 10 min. The lysis mixture was clarified at 13,000 rpm for 20 min at 4℃. The supernatant was collected as the lysate, and the protein concentration was determined by a Bradford protein assay (Sigma, St. Louis MO, USA). The 20 µg of lysate protein was mixed with 5x SDS-PAGE sample buffer containing 60 mM Tris-HCl (pH 6.8), 25% glycerol, 2% SDS, 10% bromophenol, and 5% 2-mercaptoethanol, and heated at 95℃ for 10 min, and separated by SDS-PAGE. After electrophoresis, the proteins were transferred onto a nitrocellulose membrane using a transblot chamber with Tris transfer buffer (0.025 M Tris-HCl, 0.192 M glycine, and 20% MeOH). The membrane was blocked for 1 hour at 4℃ with 5% nonfat milk in TBS-T buffer containing 10 mM Tris-HCl (pH 6.8), 100 mM NaCl, and 0.1% Tween 20. The membranes were then incubated overnight with either of the following primary antibodies from Cell Signaling Technology (Beverly MA, USA): ErbB2 and ErbB3 (all used at 1:1,000 dilutions). In addition, antibody from β-actin was from Santa Cruz Biotechnology (CA, USA) (also used 1:1,000). After washing with TBS-T buffer, the membranes were incubated with horseradish peroxidase conjugated secondary antibody (goat anti-rabbit or mouse anti-goat at 1:2,000) for 1 hour at room temperature. Bands were detected by chemiluminescent substrate (IMGENEX, San Diego CA, USA), and quantified using UVP imaging system (UVP, Upland CA, USA) and Vision Works TMLS analysis software (UVP).