Quantifying Epigenetic Modifications in AC16 Cells

AC16 cells were collected and resuspended in RIPA lysis buffer (ThermoFisher) containing a Protease and Phosphatase Inhibitor Cocktail (Roche) and analyzed as previously described (27 (link)). Briefly, samples were centrifuged, and the supernatants were collected. Protein concentration was determined by BCA assay (Sigma-Aldrich). Fifty microgram total protein was loaded on a 4–20% mini-protean TGX precast protein gel and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% skim milk and probed with the primary antibodies diluted at 1/1,000 (EZH2, Cell signaling # #5246S; GAPDH, Invitrogen, #AM4300; H3K27me3, Cell signaling, #9733S, Histone H3, Cell Signaling, #9715S) overnight at 4°C followed by corresponding secondary HRP-conjugated antibodies (Cell signaling). The blots were developed using the SuperSignal West Dura Reagent (Thermo Scientific) and visualized using a BioRad imager.