Extended Data Fig. 1: Identification of BACH1 as a specific binding partner of p53R175H.
a, Diagram of the construct of SFB-tagged wild-type p53 DBD + TD domains. DBD, DNA binding ___domain; TD, tetramerization ___domain; SFB tag: S-protein, Flag and streptavidin binding peptide (SBP). b, Silver staining of SDS–PAGE gel loaded with SFB-p53 DBD + TD protein complex purified from H1299 cell line stably overexpressing SFB-p53 DBD + TD protein by double IP (SBP IP + S-protein IP). c, BACH1 peptides sequences identified from Mass-Spec of p53 DBD + TD protein complex. d, Co-IP of SFB-tagged p53 DBD (WT, R175H, R248W, R273H, G245S, R249S and R282W) with Flag-HA (FH)-BACH1. H1299 p53 null cells were co-transfected with SFB-tagged p53 DBD and FH-BACH1 and 24 h later, cells were lysed with NP40 lysis buffer and immunoprecipitated with SBP beads, followed by Western blot analysis of BACH1 and Flag (SFB-p53). e, p53R175H-BACH1 endogenous Co-IP by BACH1 specific antibody in Cal-33 cells. f, Endogenous co-IP of p53 and BACH1 in mutant p53 cell lines MDA-MB-231 (p53R280K). g, Co-IP of SFB-tagged p53R175H truncated domains (100-300-R175H, 1-160 and 98-180-R175H) with Flag-HA (FH)-BACH1 in H1299 cells. h, Co-IP of SFB-tagged BACH1 with p53R175H and its deletion mutant (∆161-180 aa) in H1299 cells. SFB, S-protein-Flag-Streptavidin binding peptide; SBP, streptavidin binding peptide. Gel staining (b) and Western blot experiments (d-h) were repeated three times with similar results and representative images are shown.
