Ubiquitination and Androgen Receptor Analysis

22Rv1 cells co-transfected with pRBG4-CW7-myc-ubiquitin (cDNA encoding Myc-tagged human ubiquitin, kindly provided by Xiaofang Yu) were treated with or without 5 µM Z15 in the presence of 5 µM MG132 for 12 hr. Then, the cells were washed with ice-cold PBS and collected in NP-40 buffer. The lysates were lysed for 1 hr in an ice bath, then centrifuged at 12,000 g for 10 min. The supernatants were incubated with 5 µL antibody to AR (sc-7305; Santa Cruz) overnight at 4 ℃, with 40 µL protein A/G and rocked for 4 hr at 4 ℃. The protein A/G beads were pelleted and washed twice with ice-cold PBS. The precipitates were resolved on an SDS-polyacrylamide gel electrophoresis gel and subjected to Western blot analysis.