Fig. 3

ING3 promotes AR acetylation and regulates its nuclear translocation. a TIP60 was immunoprecipitated from C4-2 lysates from cells grown +/– 10 nM MB for 24 h, and lysates were blotted for TIP60 and AR. b Cells were co-transfected with AR, TIP60, and increasing amounts of ING3 plasmid + 10 nM MB for 24 h. AR or TIP60 was immunoprecipitated using α-AR or HA-affinity beads, respectively. The graph shows the average ratio of AR:HA-TIP60 in three independent experiments (t test **P < 0.01). c C4-2 cells transfected with siCtrl or siING3 were treated for 24 h with MB, immunoprecipitated with α-TIP60, and blotted with α-TIP60 or α-AR. The graph shows the average TIP60:AR ratio (t test *P < 0.05). d ARE reporter activity of cells co-transfected with 1 μg AR3-tkk-LUC, 0.2 μg β-gal, 0.2 μg AR, 50 ng of TIP60, and 20 nM of either siCtrl or siING3 for 48 h +/– 1 nM MB for the final 24 h (ANOVA *P < 0.05). e HEK293T cells were co-transfected with 1 μg of vector or AR construct +/– 0.2 μg ING3, and the AR acetylation was determined. The graph shows the average ratio of Ac-AR:total AR (t test *P < 0.05). f, g HEK293T cells were co-transfected with AR-Myc, ING3-HA, or GFP (f) or with AR-Myc, siCtrl, or siING3 (g). AR was immunoprecipitated with α-myc. ING3 was immunoprecipitated with α-HA in GFP or ING3-transfected cells (f). TIP60 was immunoprecipitated in siCtrl- or siING3-transfected cells (g). In vitro acetylation assays were performed using 1 mM of acetyl coenzyme A. h Cells were co-transfected with 1 μg AR3-tkk-LUC, 0.2 μg β-gal, 0.2 μg of either vector or ING3, and 0.2 μg of either wild-type or mutant AR constructs for 48 h +/– 0.1 nM MB for 24 h. i LNCaP cells were transfected with siCtrl or siING3 for 48 h. After 2 h of treatment with MB, nuclear and cytoplasmic fractions were blotted to detect AR. j LNCaP cells were transfected with GFP or ING3-HA under androgen deprivation conditions and stained with anti-AR and anti-HA antibodies. The nuclear intensity of AR staining for transfected cells and the adjacent untransfected cells was measured using ImageJ, and the relative ratio was graphed (t test *P < 0.05)