Fig. 7

MDM2 is recruited by SUMO3 modificd PIAS1 and required for degradation of AR. (a) DU145 cells were transiently co-transfected with flag-AR, GFP-SUMO3 and myc-PIAS1 or myc-PIAS1 (K117 L) for 72 h. Whole-cell lysates were immunoblotted with anti-AR, anti-myc and anti-actin antibodies. (b) DU145 cells were transiently co-transfected as described in A for 48 h. Whole-cell lysates were immunoprepapited with anti-myc antibodies. The immunoprepapite was then detected by indicated antibodies against myc, MDM2 and ChIP. Whole-cell lysates (Input) were also immunoblotted with anti-myc, anti-MDM2, anti-ChIP and anti-actin antibodies. (c) DU145 cells were transiently co-transfected with PIAS1, empty vector or GFP-SUMO3 or GFP-SUMO3 and myc-PIAS1. Whole-cell lysates were immunoprepapited with anti-myc antibody. The immunoprepapite was then detected by indicated antibodies against myc, SUMO3, MDM2 and ChIP. Whole-cell lysates (Input) were also immunoblotted with anti-myc, anti-MDM2, anti-ChIP and anti-actin antibodies. (d) DU145 cells were transiently co-transfected with flag-AR, myc-PIAS1 and empty vector or GFP-SUMO3 or GFP-SUMO3 and MDM2 shRNA for 72 h. Whole-cell lysates were immunoblotted with anti-AR, anti-myc and anti-actin antibodies