Western Blot Analysis of Protein Modifications

Western blot analysis was performed as described previously42 (link). Samples were collected directly in 1 × NuPAGE LDS sample buffer with 1 × Sample reducing buffer (Invitrogen) and denatured at 95 °C for 5 min followed by a centrifugation at 18,500g for 5 min. The supernatant was electrophoresed on a 4–12% Tris-HCl gel and transferred to nitrocellulose membrane (Invitrogen). After blocking with Superblock T20 blocking buffer (Thermo Scientific), the membrane was incubated with a primary antibody overnight at 4 °C and then with a secondary antibody conjugated with alkaline phosphatase (1 h at room temperature); the signal was detected by using a chemiluminescence method. The following primary antibodies were used: anti-SETDB1 (Santa Cruz, 1:1,000), anti-p53 (Cell Signaling, 1:1,000), anti-p53/K370me1 and anti-p53/K370me2 (in-house made as previously described32 (link); 1:1,000), anti-p53/K372me1 (ab16033, AbCAM, 1:1,000), anti-GAPDH (7B; Santa Cruz, 1:10,000), anti-H3K9me3 (39161, Active Motif, 1:1,000), anti-p-p53(S15; 16G8; Cell Signaling, 1:1,000) and anti-MDM2 (1F2; Millipore, 1:200).