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. 2016 Jan 31;7(20):28836–28848. doi: 10.18632/oncotarget.7089

Figure 1. ΔNp63α binds SETDB1.

Figure 1

(A) Semi-endogenous immunoprecipitation of p63α isoforms and SETDB1. Flag-TAp63α and Flag-ΔNp63α expression vectors were transiently transfected in H1299 cells. Cell extracts were immunoprecipitated with anti-Flag antibody and subjected to western blot analysis (lanes 4 to 6) with anti-SETDB1 antibody (upper panel) and anti-Flag antibody (lower panel). Aliquots of total cell extracts from unprocessed cells were also loaded on the gel (lanes from 1 to 3). EV, empty vector. (B) Immunoprecipitation of endogenous p63 with endogenous SETDB1. MCF7 cells extracts were immunoprecipitated with anti-p63 antibody and subjected to western blot analysis (lane 3) with anti-SETDB1 antibody (lower panel) and anti-p63 antibody (upper panel). The aliquot of total cell extract from unprocessed cells (lane 1) and IgG, used as negative control, (lane 2) were also loaded on the gel. Quantification of SETDB1 IP/SETDB1 IgG = 1,8 fold. (C) It is shown wt ΔNp63α containing all domains of the protein: Transactivation domain of ΔN isoforms (TA1, not shown), DNA-binding domain (DBD), oligomerization domain (OD), transactivation domain 2 (TA2, not shown), sterile alpha motif (SAM) and transactivation inhibitory domain (TID); the first mutant contains only the C-terminus including OD, TA2 (not shown), SAM, TID (CT); the second one contains all domains apart from TID (ΔTID); the third one contains all domains apart from SAM and TID (ΔSAM-TID); the fourth one contains all domains apart from OD, TA2, SAM, TID (NT). (D) Coimmunoprecipitation of p63 deletion mutants and SETDB1. HA-ΔNp63α, HA-CT, HA-ΔTID, HA- ΔSAM-TID, HA-NT expression vectors were transiently transfected in H1299 cells together with full lenght Flag-SETDB1. Cell extracts were immunoprecipitated with anti-SETDB1 antibody and subjected to western blot analysis (lanes 7 to 12) with anti-SETDB1 antibody (upper panel) and anti-HA antibody (lower panel). Aliquots of total cell extracts from unprocessed cells were also loaded on the gel (lanes 1 to 6). EV, empty vector; NT, no transfection. (E) It is shown wt SETDB1 containing all domains of the protein: Tudor domains (TUD), methyl-CpG-binding domain (MBD), PRE-SET domain and the bifurcated SET domain (S-ET). Post-SET domain is not shown; the first mutant (1-256 aa) contains only the N-terminus, lacking all functional domains; the second mutant (1- 615 aa) contains the N-terminus and both Tudor domains; the third mutant contains MBD (methyl-CpG-binding domain), PRE-SET and the bifurcated SET domain. (F) Coimmunoprecipitation of SETDB1 deletion mutants and ΔNp63α. Full lenght Flag-SETDB1, Flag-1-256aa, Flag-1-615aa and Flag-528-1307aa expression vectors were transiently transfected in H1299 cells together with full lenght ΔNp63α. Cell extracts were immunoprecipitated with anti-Flag antibody and subjected to western blot analysis (lanes 6 to 10) with anti-Flag antibody (upper panel) and anti-HA antibody (lower panel). Aliquots of total cell extracts from unprocessed cells were also loaded on the gel (lanes 1 to 5). EV, empty vector. Uncropped images of gels are shown in Supplementary Figure S5.