Fig. 2: Acetylation on K382 of p53 promotes PBRM1 binding.
From: PBRM1 acts as a p53 lysine-acetylation reader to suppress renal tumor growth
a Schematic depiction of the functional domains of p53 and truncated constructs. FL full-length, TAD transactivation domain, PRD proline-rich domain, DBD DNA-binding domain, TD tetramerization domain, CTD C-terminal domain. b HEK293T cells were transfected with vectors, Flag-PBRM1, Myc-p53 and Myc-p53 truncated constructs. After immunoprecipitation with Flag-M2 beads, the inputs and eluates were analyzed by immunoblots. c Vectors, Flag-PBRM1, Myc-p53 and HA-Tip60, HA-PCAF, HA-CBP or HA-p300 were transfected into HEK293T cells as indicated. Lysates were used for anti-Flag immunoprecipitation and 3X Flag peptide elution. The inputs and eluates were analyzed by immunoblots. d HEK293T cells were transfected with Flag-PBRM1, Myc-p53 and HA-p300. The amount of HA-p300 was titrated as indicated. Lysates were used in immunoprecipitations with Flag-M2 beads, and inputs and eluates were analyzed by immunoblots. e Vectors, Flag-PBRM1, HA-p300 and Myc-p53 (WT) or Myc-p53 6KR mutant were transfected into HEK293T cells as indicated. Lysates were used for anti-Flag immunoprecipitation and 3X Flag peptide elution. Inputs and eluates were examined by immunoblots. 6KR: lysines mutated to arginines on p53 at K370, K372, K373, K381, K382, and K386. f, g Biotinylated p53 peptides with lysine acetylation at the indicated sites were incubated with lysates from H1299 cells (f) or H1299 cells transfected with full-length PBRM1 or the PBRM1 bromodomains (Flag-PBRM1 FL or Flag-PBRM1-BD1–6, respectively, g). The peptides were pulled down with streptavidin beads and the associated proteins were analyzed by immunoblots. 6KAc: lysines acetylated on p53 at K370, K372, K373, K381, K382, and K386. h Vectors, Flag-PBRM1, HA-p300 and Myc-p53 (WT) or Myc-p53 K382R mutant were transfected into HEK293T cells as indicated. Lysates were used for anti-Flag immunoprecipitation and 3X Flag peptide elution. Inputs and eluates were examined by immunoblots. Source data are provided as a Source Data file.
