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. 2020 Dec 22;49(2):902–915. doi: 10.1093/nar/gkaa1224

Figure 3.

Figure 3.

USP7 interacts with and targets ubiquitylated SPRTN in human cells. (A) Analysis of DUB overexpression-induced deubiquitylation of endogenous SPRTN in HeLa T-REx Flp-In cells. Indicated N-terminally Flag-tagged DUBs were transiently expressed for one day before cells were lysed and analysed by western blotting. (B) Analysis of DUB overexpression-induced deubiquitylation of doxycycline-inducible YFP-SPRTN-Strep stably expressed in HeLa T-REx Flp-In cells. Indicated N-terminally Flag-tagged DUBs were transiently expressed for one day before cells were lysed and analysed by western blotting. (C) Increasing amounts of N-terminally Flag-tagged USP7 (or the catalytically inactive CS variant) were transiently expressed in HeLa T-REx Flp-In cells for one day before cells were lysed and analysed by western blotting. (D) Increasing amounts of N-terminally Flag-tagged USP7 (or the catalytically inactive CS variant) were transiently expressed in HeLa T-REx Flp-In cells stably expressing doxycycline-inducible YFP-SPRTN-Strep for one day before cells were lysed and analysed by western blotting. (E) Plasmids encoding Flag-tagged full-length USP7 (WT or the catalytically inactive CS variant) were transiently transfected in HeLa T-REx Flp-In cells stably expressing the indicated doxycycline-inducible YFP-SPRTN-Strep variants. Binding was analysed by co-immunoprecipitation using anti-Flag beads followed by western blotting. (F) Schematic depiction of USP7’s domain structure and protein truncations used for co-immunoprecipitation analysis with SPRTN (upper panel). Plasmids encoding Flag-tagged full-length USP7 (WT or the catalytically inactive CS variant) or the respective truncations were transiently transfected in HeLa T-REx Flp-In cells stably expressing doxycycline-inducible YFP-SPRTN-Strep. Binding was analysed by co-immunoprecipitation using anti-Flag beads followed by western blotting (lower panel).