Fig. 5

CircWSB1 directly binds with deubiquitinase USP10. a Silver staining of proteins pulled down by biotin-labeled probe specific for circWSB1 and control probe. b The RNA pull-down proteins identified by mass spectrometry analysis. c Unique peptides of USP10 identified by mass spectrometry analysis. d The interaction between circWSB1 and USP10 was verified by western blot. e FISH and IF co-staining indicating the co-localization of circWSB1 (red) and USP10 (green) in MCF-7 and MDA-MB-453 cells cultured under hypoxia. Scale bar, 50 μm. f and g RIP assays were carried out in hypoxic MCF-7 cells under indicated conditions using anti-USP10 and IgG control, followed by qRT-PCR. h Interaction profile of circWSB1 on USP10. i Heatmap of interaction between circWSB1 and N-terminal region (1-100aa) of USP10. j RNA EMSA showing the interaction between USP10 protein and biotin-labeled probe of circWSB1. k Schematic diagram of full-length and truncated USP10 protein. l RIP assays were executed with anti-Flag in hypoxic MCF-7 cells transfected with indicated full-length or truncated USP10 plasmids with 3 × Flag. Co-precipitated proteins and RNAs were purified and followed by western blot and qRT-PCR, respectively. m RNA pull-down assays using biotin-labeled circWSB1 probe in hypoxic MCF-7 cells expressing full-length of USP10 and its deletion mutants. The pulled down proteins were subjected to western blot. Data are shown as mean ± SD and representative of three independent experiments in (f-g) and i. **P < 0.01, ***P < 0.001