Western Blot Protein Analysis Protocol

Total protein extracts were prepared using a lysis buffer (50 mM Tris, 2% SDS, 10% glycerol, 0.74 M beta-mercaptoethanol), sonicated on ice using a Sonifier 250D (Branson Ultrasonics, St. Louis MO, USA), and heated for 5 min at 99 °C. Protein concentrations were determined with the Bio-Rad DC Protein Assay Kit (BioRad, Hercules, CA, USA) using bovine serum albumin as a standard. Protein extracts were separated in 10% SDS-PAGE, electrotransferred to PVDF membranes (Immobilon-P; EMD Millipore, Burlington, MA, USA). Antibodies used are: HSP90 antibody [H90-10] #ab53497, MYC #ab32 (Abcam, Cambridge, UK) [36 (link)], #sc764 (Santa Cruz Biotechnology, Dallas, TX, USA) [37 (link)]; TUBULIN #ab6046 (Abcam, Cambridge, UK) [38 (link)]; Anti-MOUSE HRP #31430 (Thermo Fisher, Waltham, MA, USA) [39 (link)]; Anti-RABBIT HRP #31460 (Thermo Fisher, Waltham, MA, USA) [40 (link)].