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. 2021 Jan 4;131(1):e134565. doi: 10.1172/JCI134565

Figure 2. USP7 preferentially interacts with AR112Q in cells.

Figure 2

(A) Co-IP of USP7 with AR after pull-down with an anti-AR antibody, followed by immunoblot with anti-AR or anti-USP7 antibodies; GAPDH detection served as loading control. Input levels of AR or USP7 in AR112Q- relative to AR10Q-expressing cells are shown in blue (data normalized to AR10Q cells). Relative amounts of immunoprecipitated AR or USP7 are shown in red, normalized to IP recovery from AR10Q-expressing cells. (B) Co-IP of AR with USP7 following pull-down with an anti-USP7 antibody, followed by immunoblot with anti-AR or anti-USP7 antibodies; GAPDH detection served as loading control. As in A, relative input levels are shown in blue, and amounts of immunoprecipitated proteins are shown in red. (C) USP7-AR interaction was evaluated by PLA (red puncta) in PC12 cells. AR was predominantly nuclear, as judged by subsequent immunostaining with anti-AR antibody (green signal). Scale bar: 10 μm. (D) Quantification of PLA puncta in cells expressing AR10Q and AR112Q, respectively, based on images taken before staining for total AR (see Supplemental Figure 2C), with 100 cells evaluated per condition and carried out in triplicate. P < 0.0001, Kolmogorov-Smirnov test.