Protein Extraction and Western Blot Analysis

Whole cell extracts were prepared by lysing the cells or emboli with RIPA buffer (25 mM Tris [pH 8.0]; 50 mM NaCl; 1 mM EDTA; 0.5% NP40; 0.5% sodium deoxycholate; 0.1% SDS; 10 μL/mL protease inhibitor cocktail, MilliporeSigma, P8340; 10 μL/mL phosphatase inhibitor cocktail #2, MilliporeSigma, P5726; 10 μL/mL phosphatase inhibitor cocktail #3, MilliporeSigma, P0044). Tumor extracts were prepared as described (27 (link)). Protein concentrations were measured by BCA assay (Thermo Fisher Scientific, 23225). About 10–20 μg protein was loaded onto NOVEX WedgeWell 4%–20% Tris-glycine gels, and Western blot analyses were carried out as described (79 (link)). Representative data are shown and were repeated at least 3 times each.