Immunoprecipitation of SETDB1 and p53

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.