Figure 2. VCPIP1 deubiquitinates SPRTN and is important for SPRTN relocalization to chromatin upon DPCs.

(A) U2OS cells were treated for 2 h with the indicated dose of FA and Western blots were performed with the indicated antibodies.

(B) Control (Ctrl) or VCPIP1-knockdown U2OS cells were treated with or without FA (2 mM, 2 h) and Western blots were performed with the indicated antibodies.

(C) Control (Ctrl) or VCPIP1-knockdown HEK293T cells were transfected with vector (Vec), WT or C218A (CA) Flag-VCPIP1 and treated with FA (2 mM, 2 h). Western blots were performed with the indicated antibodies.

(D) Flag-SPRTN isolated from cells was incubated with GST-tagged purified wild-type (WT) VCPIP1 or catalytically inactive (CA) VCPIP1 in vitro and immunoblotted with the indicated antibodies.

(E) Control or VCPIP1-knockdown U2OS cells were treated with FA (2 mM, 2 h). Cells were either lysed directly in SDS-containing loading dye (total) or subjected to fractionation into soluble and chromatin components. Western blots were performed with the indicated antibodies.

(F) VCPIP1-knockdown HEK293T cells were transfected with vector (Vec), wild-type (WT) or C218A (CA) Flag-VCPIP1 and treated with FA (2 mM, 2 h). Cells were lysed as in (E) and immunoblotted with the indicated antibodies.

(G-H) VCPIP1-knockdown U2OS cells stably expressing vector (Vec), WT or C218A (CA) Flag-VCPIP1 were treated with CPT (1 μM, 30 min). Top1cc signals were detected by immunofluorescence. Nuclei were visualized with DAPI (blue). Representative images are shown in (G). Quantification of foci signals is shown in (H). Error bars indicate mean ± SEM of three independent experiments (** p<0.01; ns: not significant). Scale bars, 10 μm.

See also Figure S2.