FIG. 8.

ARIP3 and PIAS1 enhance the sumoylation of AR in HeLa cells. HeLa cells seeded onto six-well plates were cotransfected with 100 ng of pcDNA-Flag-hAR; 100 ng of pFLAG-CMV2, pFLAG-ARIP3, or pFLAG-PIAS1; and 0, 10, or 50 ng of pSG5-His-SUMO-1. Cells were lysed in RIPA-1 buffer, and the lysates were immunoblotted with the anti-AR antibody. The difference between the migration of AR forms sumoylated with endogenous SUMO-1 and that of ectopically expressed SUMO-1 is due to the His tag present in the SUMO-1 expression vector. The identities of sumoylated AR forms were verified by immunoblotting the anti-AR antibody-immunoprecipitated AR with the anti-SUMO-1 antibody (not shown).