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

Figure 3. PP1α engages predominantly with the AR ligand-binding domain (LBD) that is enriched in ubiquitination.

Figure 3

A. 293T cells in medium containing 5% FBS were transfected with Flag-tagged PP1α (Flag-PP1α) and HA-tagged AR full length (HA-AR FL) versus additional AR constructs (N: NTD; N-D: NTD-DBD; D: DBD; D-L: DBD-LBD; and L: LBD), followed by anti-Flag co-IP analysis for blotting. Arrows indicate native AR bands and non-specific IgG bands are marked (*). Flag-tagged Sox9, which weakly interacts with AR, was used as control. B. Similarly, 293T cells were transfected with Flag-PP1α and HA-AR full length versus NTD-DBD and LBD constructs. After transfection, cells were then incubated for overnight in androgen-depleted medium for co-IP and blotting. C. 293T cells in androgen-depleted medium were transfected with His-Ub, Flag-PP1α, and HA-AR constructs as indicated, followed by Ni(nickel)-NTA-IP under denatured condition and blotted. D. Similarly, 293T cells in androgen-depleted medium were transfected with His-Ub and indicated HA-AR constructs, followed by Ni-NTA-IP under denatured conditions. Arrowheads in the upper panels mark positions of the native AR constructs and an arrowhead on the lower panel of (C) marks the mono-ubiquitylated PP1α. Mr: Molecular weight marker in Kilodalton (KDa).