Cdkn2a p16

Cell lysates were prepared using RIPA buffer (Sigma)/Halt protease inhibitor cocktail (Thermo Fisher, Rockford, IL) and the protein concentrations were determined using the BCA protein assay (Thermo Fisher). Equal protein amounts were subjected to SDS-PAGE and transferred onto PVDF membranes (Bio-Rad). Immunodetection was performed using the following primary antibodies: BIM, BMI1, DNMT1, EZH2, Histone3, β-actin (Cell signaling), H3K9me3, H3K27me3, CDKN2A (p16) (abcam) and the appropriate ECL HPR-linked secondary antibodies (GE healthcare).

Protein was extracted from untreated PDCLs using cell lysis buffer (10 mM Tris-Cl pH 7.4, 100 mM NaCl, 1 mM EDTA pH 8.0, 1 mM NaF, 20 mM Na₄P₂O₇, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 10% Glycerol, Milli-Q water) and cOmplete, mini, EDTA-free protease inhibitor tablets (Sigma-Aldrich). Western blots were probed with antibodies against CDK4 (Cell Signaling, Danvers, MA, USA; 1 : 2000); CDK6 (Cell Signaling; 1 : 2000); phosphorylated RB1 (Cell Signaling; 1 : 1000); E2F (Cell Signaling; 1 : 1000); RB1 (Abcam, Cambridge, MA, USA; 1 : 1000); CDKN2A/p16 (Abcam; 1 : 1000); gamma histone H2A.X (H2AX; Cell Signaling; 1 : 1000); cleaved PARP (Cell Signaling; 1 : 1000). To control for protein loading, membranes were probed with alpha-tubulin (Abcam; 1 : 10 000).