Fig. 3

HBXIP enhances acetylation of HOXB13 at K277 site via acetylase p300. a Immunoblotting analysis of HOXB13 in MCF-7 cells time-dependently treated with 100 μg/ml cycloheximide (CHX) after being transiently transfected with the indicated plasmids. b Immunoblotting analysis of exogenous Flag-HOXB13 in HEK293T cells. The cells were transiently transfected with pCMV-HOXB13 accompanied by pcDNA or pcDNA-HBXIP. The protein level of Flag-HOXB13 was examined by the anti-Flag antibody. c Acetylation level of HOXB13 was detected by a Co-IP assay using GFP-beads and an immunoblotting analysis performed with the anti-acetyl-lysine antibody in HEK293T cells. The cells were transiently transfected with the indicated plasmids. The protein levels of HBXIP and HOXB13 were examined by anti-Flag or anti-HOXB13 antibodies, respectively. The left and right panels are identical results that differ in exposure time. d, e Immunoblotting analysis of HOXB13 in MCF-7 cells and HEK293T cells treated with 100 μg/ml CHX along with different concentrations of trichostatin A (TSA) for 18 h (d) or 1 μM TSA (e) for the indicated time points. f Acetylation level of HOXB13 was detected by a Co-IP assay using GFP-beads and an immunoblotting analysis performed with the anti-acetyl-lysine antibody in HEK293T cells. The cells were separately transiently transfected with GFP-HOXB13-WT, GFP-HOXB13-K270R, GFP-HOXB13-K277R, or GFP-HOXB13-DM along with pCMV or pCMV-HBXIP. The protein levels of HBXIP and HOXB13 were examined by anti-Flag or anti-GFP antibodies, respectively. g Acetylation level of HOXB13 was detected by a Co-IP assay using GFP-beads and an immunoblotting analysis with the anti-acetyl-lysine antibody in HEK293T cells. The cells were transiently transfected with GFP-HOXB13-WT along with the indicated plasmids or siRNAs. The protein levels of HBXIP and HOXB13 were separately examined by anti-Flag or anti-GFP antibodies. All experiments were repeated at least three times