P21 epr362

Protein extraction and western blot were performed as previously described [41 (link), 46 (link)]. Antibodies used are as follows: Ubqln4 (A333, 1:1000, Santa Cruz, CA, USA); p53 (DO-1, 1:1000, Abcam, Cambridge, UK); p21 (EPR362, 1:1000, Abcam); RNF114 (SM-3, 1:1000, Santa Cruz); Flag (3B9, 1:5000, Abmart, Shanghai, China); HA (C29F4, 1:1000, CST); GAPDH (D16H11, 1:3000, CST); and β-actin (8H10D10, 1:1000, CST).

Cells were washed with ice-cold PBS and lysed in ice-cold RIPA lysis buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 0.5% TritonX-100, 0.5 mM EDTA, pH 8) supplemented with protease inhibitor cocktails (Sigma) and phosphatase inhibitor cocktail (Roche, Sigma). Lysates were cleared by centrifugation at 15 000 rpm for 10 min at 4°C; supernatants were removed and assayed for protein concentration using the Quick Start™ Bradford Protein Assay Kit (Bio-Rad). Lysates were denatured by addition of 5× Laemmli sample buffer (1% sodium dodecyl sulfate, 300 mM Tris-HCl pH 8, 50% glycerol, 0.025% bromophenol blue and 10% 2-mercaptoethanol) and incubation for 5 min at 95°C. At least 20 μg were subjected to SDS-PAGE and transferred to PDVF membranes (Bio-Rad), following the manufacturers’ instructions. The following antibodies were used: RAS (3965, CST), MYC (clone Y69, Abcam), AKT (9272 CST), DKC1 (H-300, Santa Cruz Biotechnology), ATRX (H-300, Santa Cruz Biotechnology), ATRX N-terminal (D1N2E clone, CST), p21 (EPR362, Abcam), p53 (DO-1, Santa Cruz Biotechnology), Phospho-p53 Ser46 (2521 CST), HIPK2 (5091, CST) and β-actin (clone A-15, Sigma-Aldrich).