Mabe305

Total protein extract was obtained using RIPA buffer. Samples were subjected to electrophoresis in fast cast SDS-PAGE gels according to the manufacturer’s protocol (Bio-Rad). Next, the separated proteins were transferred onto an TransBlot Turbo Polyvinylidene Fluoride (PVDF) membrane using a Trans-Blot Turbo Transfer System (Bio-Rad). PVDF membranes were imaged on the Bio-Rad ChemiDoc imaging system to verify transfer. Membranes were blocked in EveryBlot Blocking Buffer for 5 min at room temperature and then incubated overnight at 4 °C with the primary monoclonal antibody, anti-DNMT3B (1:2000; MABE305, Millipore). The membranes were then incubated for 1 h at room temperature with secondary antibody horseradish peroxidase-conjugated anti-rat IgG (1:5000; AP136P, Millipore). After incubation, the blots were imaged on a Bio-Rad ChemiDoc imaging system in order to measure total protein content as a loading control for protein normalization, as previously described [36 (link)]. Membranes were developed with the Clarity Western ECL Substrate (Bio-Rad), chemiluminescent signals were acquired on ChemiDoc MP (Bio-Rad) and band intensities were analyzed using Image Lab software (Bio-Rad).