Fig. 3
From: Rolapitant treats lung cancer by targeting deubiquitinase OTUD3
Rolapitant inhibits the deubiquitinating activity of OTUD3. A, B Western blot analysis of GRP78 and OTUD3 levels in A549 and H1299 cells treated with DMSO or indicated concentrations (0–25 μM) of Rolapitant for 48 h. GAPDH was used as a loading control. C OTUD3 was knocked down by shRNA in H1299 cells. H1299 cells were treated with 10 μM Rolapitant for 48 h, and cell lysates were assessed by western blot. D HEK293T cells were transfected with HA-Ub, Myc-GRP78, Flag-OTUD3, Flag-OTUD3C76A alone or in combination and incubated with or without 5 μM Rolapitant for 46 h. Proteasome inhibitors (20 μM MG132) were added 6 h before lysis. Cell lysates were immunoprecipitated with anti-Myc antibody, followed by western blot with indicated antibodies. E OTUD3 OTU (aa 52–209) and K48-linked Di-Ubiquitin were incubated with 10 μM Rolapitant for 3 h at 37 °C, followed by western blot with indicated antibodies. F Flag-OTUD3 and Myc-GRP78 were co-transfected into HEK293T cells with indicated concentrations of Rolapitant for 24 h. Cell lysates were subjected to immunoprecipitation with anti-Flag antibody, followed by western blot with indicated antibodies. G Flag-OTUD3 and Myc-OTUD3 were co-transfected into HEK293T cells with indicated concentrations of Rolapitant for 24 h. Cell lysates were subjected to immunoprecipitation with anti-Flag antibody, followed by western blot with indicated antibodies. H HEK293T cells transfected with Myc-GRP78 were lysed and lysates incubated with GST or GST-OTUD3 proteins with increasing concentrations of Rolapitant treatment for 4 h. Proteins retained on Sepharose were blotted with the indicated antibodies, or visualized by SDS-PAGE and Coomassie blue staining. I Purified GST-OTUD3 and GST proteins were incubated with extracts from Flag-OTUD3 transfected HEK293T cells with increasing concentrations of Rolapitant treatment for 4 h. Proteins retained on Sepharose were blotted with the indicated antibodies, or visualized by SDS-PAGE and Coomassie blue staining. All panels are representative results of three or more independent experiments