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. 2015 Nov 30;7(2):1754–1764. doi: 10.18632/oncotarget.6434

Figure 4. PP1α stabilizes AR by attenuating LBD-directed AR ubiquitylation.

Figure 4

A. 293T cells transfected with His-Ub, HA-AR-LBD and Flag-PP1α were incubated overnight in androgen-depleted medium without or with MG132 (10 μM) as indicated, followed by Ni-NTA-IP under denatured conditions and blotting. SE: short exposure; LE: long exposure. B. 293T cells were transfected with HA-Ub, Flag-AR-full-length together without or with PP1α. Cells were then incubated overnight in androgen-depleted medium, followed by anti-Flag-IP under denatured conditions. Mr: Molecular weight marker in Kilodalton (KDa). C. HeLa cells in androgen-depleted medium were co-transfected with HA-AR full-length (FL), NTD-DBD and LBD constructs, without or with Flag-PP1α and cycloheximide as indicated. Total proteins were harvested and normalized for blotting. D. HeLa cells in androgen-depleted medium were co-transfected with AR and PP1α wild-type versus its enzymatically inactive counterpart (H248K). Cells were then incubated in androgen-depleted medium for blotting.