Precision plus protein kaleidoscope molecular

Cells or homogenized tumor specimens were lysed using radio-immunoprecipitation assay (RIPA) buffer supplemented with protease inhibitors (Sigma Aldrich), phosphatase inhibitors (Sigma Aldrich), and phenyl-methane-sulfonyl-fluoride (Sigma Aldrich). Immunoblotting, gel transfer, and immunodetection was performed as previously described (4 (link)). The Precision Plus Protein Kaleidoscope molecular weight marker (Bio-Rad) was used to confirm the expected size of target proteins. Antibodies were utilized according to the manufacturers’ recommendations. Equal protein loading was confirmed using vinculin, GAPDH or β-actin.

Whole-cell lysates were isolated using radioimmunoprecipitation (RIPA) buffer supplemented with protease inhibitor (Sigma), phosphatase inhibitor (Sigma), and phenylmethane-sulfonylfluoride (PMSF) (Sigma) as previously described (15 (link)). Protein concentrations were determined using a Micro BCA Protein Assay Kit (Thermo Fisher Scientific Inc.), and samples were separated by electrophoresis on sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gels. The Precision Plus Protein Kaleidoscope molecular weight marker (Bio-Rad, Hercules, CA) was utilized to confirm expected size of target proteins. Gel transfer to blotting membrane was completed using Trans-Blot Turbo Transfer System (Bio-Rad) per manufacturer’s protocol. Antibodies were utilized according to the manufacturers’ recommendations. Immunoblots were developed with Immobilon Classico or Crescendo Western HRP Substrate (EMD Millipore, Millipore Sigma, Burlington, MA). Blots were stripped with stripping solution (Bio-Rad) at 65°C for 20 minutes and then re-probed with selected antibodies. Equal protein loading between samples was confirmed using β-actin as an internal control.