Figure 5. ARIP4 sumoylation mutants in AR-dependent transactivation and DNA binding.

(a) COS-1 cells were transiently transfected with 150 ng of pARE₄-tk-LUC, 20 ng of pSGhAR and 30 ng of pFLAG-ARIP4wt or indicated sumoylation mutants of ARIP4. Testosterone (T; 100 nM) was added 24 h after transfection, as depicted by the+signs, and the cells were harvested 24 h later. The values are shown relative to that of AR in the presence of testosterone without ARIP4 expression plasmids (=100). Each bar represents the mean±S.E.M. values for three independent experiments. (b) Wild-type ARIP4 or indicated sumoylation mutants of ARIP4 were produced in COS-1 cells and purified by affinity chromatography. The proteins were incubated with a ³²P-labelled 32 bp DNA fragment and protein–DNA complexes were resolved by EMSA. Asterisks indicate ARIP4-specific shifted bands.