Western Blot Analysis of Cell Lysates

Western blot assay was performed following the protocol as previously described [24 (link), 25 (link)]. Briefly, the cells were harvested and lysed using ice-cold RIPA lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM sodium chloride, 1% Nonidet P-40, and 0.5% sodium deoxycholate). Following centrifugation at 10,000×g for 15 min at 4 °C, the proteins in the supernatants were quantified by Bradford method and separated using 10% SDS-PAGE gel and electrotransferred from the gel to a nitrocellulose membrane (Merck & Co., Inc., Whitehouse Station, NJ, USA). Following blocking with 5% skimmed milk in phosphate-buffered saline, the membranes were immunoblotted with the primary antibodies, namely, CDK6 (GeneTex, San Antonio, Texas, USA), DOT1L, H3, H3K79me2, cyclin D3, and GAPDH (Abcam, Cambridge, UK) at 4 °C overnight. Specifically bound HRP-conjugated secondary antibodies were detected using an ECL detection system (ChemiDocTM XRS+ machine, Bio-Rad Laboratories). Protein levels of GAPDH and H3 were employed as loading controls. Densitometric analyses were performed using ImageJ software. Relative quantification was carried out after normalization to the band intensities of GAPDH or H3. A Mann-Whitney test was performed to assess the difference of protein expression between groups. Each experiment was performed in triplicate and repeated at least 3 times.