Anti setdb1 antibody

Immunoprecipitation after formaldehyde crosslinking was performed as previously described41 (link) with minor modifications. Briefly, cells were treated with 1% formaldehyde for crosslinking for 10 min at room temperature. Cells were harvested and lysed in 1 × RIPA buffer. The samples were sonicated until the lysate became clear, followed by centrifugation at 15,000g for 15 min at 4 °C. The supernatant was collected for immunoprecipitation (IP) using antibodies against SETDB1 (H-300; Santa Cruz, 1:250) and p53 (7F5; Cell Signaling, 1:500). The magnetic protein G beads after IP were washed three times with 1 × RIPA buffer and proteins were eluted with 2 × SDS-loading buffer. The samples were then incubated at 99 °C for 20 min before sample loading for SDS–PAGE. For non-crosslinking p53 immunoprecipitation, it was performed similarly except for the crosslinking procedure. Cells were transient transfected with wild-type p53 or p53R249S mutants. Forty-eight hours after transfection, cells were harvested and processed for IP using the Direct IP Kit (Pierce). For endogenous SETDB1 complex isolation with IP, the Nuclear Complex Co-IP Kit (Active Motif) was used for the procedure, with anti-SETDB1 antibody (Santa Cruz, 1:250) being added to the IP reaction.

Cultured MEFs were washed with cold PBS buffer and then lysed with KALB lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.5, 1 % (v/v) Triton X-100, 1 mM EDTA pH 7.5) supplemented with 1 mM sodium vanadate, 1 mM PMSF and protease inhibitors (complete mixture tablets; Roche) on ice for 30 min. Insoluble material was removed by centrifugation at 15,000 g at 4°C for 5 min. Total protein concentration in the whole cell extract was quantified using the BCA protein assay kit (Pierce) following manufacturer’s instructions. Proteins were resolved by 4–12 % SDS-PAGE (Invitrogen), transferred to PVDF membranes (Osmonics; GE) and blocked with 5 % (w/v) skim milk powder in 0.1 % (v/v) Tween-PBS for 1 h at room temperature. Membrane was incubated overnight with anti-Setdb1 antibody (1:2000 diluted; Santa Cruz, sc-66884) and anti-Actin antibody (1:2000 diluted; Santa Cruz, sc-1616), anti-H3K27me3 antibody (1:2500; Millipore 07-449), anti-H3K9me2 antibody (a:1000; Abcam, ab1220), anti-H3K9me3 (1:1000; Millipore, 07-442) at 4 °C followed with horseradish peroxidase (HRP)-conjugated secondary antibodies. Membrane was visualised using ECL system (Immobilon; Millipore) following manufacturer’s instructions.