2.7. Protein Extraction and Evaluation by Western Blot Analysis

MCF10A cells were seeded on 6 cm plastic Petri dishes (500,000 cells) and cultured as a monolayer. After 4 days of culture, they were washed, trypsinized, and collected. MCF10A cells grown in anchorage-independent conditions (10,000 cells/well) were washed and collected after 14 days of culture. Both adherent and non-adherent MCF10A cells were resuspended in RIPA buffer supplemented with a protease inhibitor cocktail (P8340, Sigma-Aldrich, Saint Louis, MO, USA, 1:100) and Na₃VO₄ (1 mM). Then, 65 μg of protein extracts were resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (AmershamTM ProtranTM Premium 0.45 μm 300 mm × 4 m). Next, the membrane was blocked for 60 min using TBS-T (0.1% Tween-20) supplemented by 3% BSA (Sigma-Aldrich, Saint Louis, MO, USA), and incubated overnight (4 °C) with the following primary antibodies: anti-ERBB2 monoclonal antibody (1:1000 dilution; #4290 Cell Signaling Technology, Inc., Danvers, MA, USA), anti-ERBB3 (1:1000 dilution; #4754 Cell Signaling Technology, Inc., Danvers, MA, USA), anti-GAPDH (1:1000 dilution; #G9545 Sigma-Aldrich, Saint Louis, MO, USA). For protein detection, the membrane was incubated with anti-rabbit horseradish peroxidase-labeled secondary antibody (Dako EnVision+ System- HRP Labelled Polymer) followed by a chemiluminescent reaction (Clarity Western ECL Substrate, Bio-Rad). Signals and images were acquired by Chemi Doc™ XRS 2015 (Bio-Rad Laboratories, Hercules, CA, USA), and densitometric analysis was performed using Image Lab software (version 5.2.1; Bio-Rad Laboratories, Hercules, CA, USA). The original western blot figures can be found in Figure S9.