Fig. 5

The formation of AR and sumoylated PIAS1 complex. (a/b) DU145 cells were cotransfected with plasmids as indicated for 48 h and the total amount of plasmids per well was normalized by empty vectors. Whole-cell lysates were immunoprepapited with anti-AR or anti-myc antibodies. The immunoprepapite was then detected by indicated antibodies against AR (IP, top panel of a) and anti-PIAS1 (IB, second panel of a), or against myc (IP, top panel of b) and anti-SUMO3 (IB, second panel of b). Whole-cell lysates (Input) were also immunoblotted with anti-AR (third panel of a), anti-myc (fourth panel of a) and anti-actin (bottom panel of a) antibodies, or with anti-myc (third panel of b), anti-AR (fourth panel of b) and anti-actin (bottom panel of b) antibodies. (c) The mammalian two-hybrid assay was performed in DU145 cells. Cells were transiently transfected in 48-well with 100 ng 5 × GAL4-luc, 25 ng Renilla luciferase reporter,30 ng SUMO3, 30 ng VP16-AR and 30 ng GAL4-PIAS1 or GAL4-PIAS1 (K117 L) as indicated. The total amount of plasmids per well was normalized in all transfections by the addition of empty vectors. Transfected cells were grown for 48 h and then harvested for luciferase assay. Values represent mean ± S.D. *P < 0.01. (d) DU145 were transiently transfected in 48-well with 100 ng 5 × GAL4-luc, 25 ng Renilla luciferase reporter,30 ng SUMO3, 30 ng GAL4-PIAS1, and 30 ng VP16-AR or various points mutants of VP16-AR constructs as indicated for 48 h, and then harvested for luciferase assay. Values represent mean ± S.D. *P < 0.05,**P < 0.01