Western Blot Analysis of Protein Extracts

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), and heated for 5 min at 99 °C. Protein concentrations were determined with the Bio-Rad DC Protein Assay Kit (BioRad) using bovine serum albumin as a standard. Protein extracts were separated in 10% SDS-PAGE and electrotransferred to PVDF membranes (Immobilon-P; EMD Millipore). Antibodies used were as follows: MYC #ab32 (Abcam, Cambridge, USA) [56 (link)], #sc764 (Santa Cruz Biotechnology, USA) [57 (link)]; DNMT3B #ab122932 (Abcam, Cambridge, USA) [58 (link)],#nb100-56514 (Novus Biologicals, USA) [59 (link)]; TUBULIN #ab6046 (Abcam, Cambridge, USA) [22 (link)]; β-ACTIN #A5441 (Sigma-Aldrich, USA); GAPDH #sc25778 (Santa Cruz Biotechnology, USA) [60 (link)]; Anti-MOUSE HRP #31430 (Thermo Fisher, USA) [61 (link)]; Anti-RABBIT HRP #31460 (Thermo Fisher, USA) [62 (link)]. Protein quantitation was performed using ImageJ software (https://imagej.nih.gov/ij/) [63 (link)].