Figure 5.
USP45 interacts with and deubiquitinates Coro1B. (A) Coimmunoprecipitation (Co-IP) analysis of the interaction between Myc-USP45 and Flag-Coro1B or Flag-Coro1C in HEK293T cells. Co-IP experiments were performed using an anti-Myc antibody, followed by a Western blot analysis with the indicated antibodies. (B) (top). Schematic presentation of domain structures and deletion mutants of Coro1B. β-propeller, β-propeller domain; CC, coiled-coil domain. (bottom) Coimmunoprecipitation assays to map the interaction regions between USP45 and Coro1B. (C) Western blot analysis of the expression levels of Coro1B and Coro1C with indicated antibodies in control and USP45 knockdown HeLa cells. (D) Quantification of Coro1B and Coro1C expression normalized to tubulin and GAPDH, respectively. Data are shown as mean ± SEM of at least three independent experiments. (E) Western blot analysis of the expression levels of Coro1B in control (scr) and USP45 knockdown HeLa cells with or without treatment of proteasome inhibitor MG132 (5 μM, 4 h). (F) Quantification of Coro1B expression normalized to GAPDH in E. Data are shown as mean ± SEM of four independent experiments. (G) Cycloheximide (CHX) chase analysis of Coro1B expression in control and USP45 knockdown HeLa cells treated with CHX for the indicated times. (H) Quantification of Coro1B levels normalized to GAPDH at different time points after CHX treatment, as indicated in G. Data shown as mean ± SEM of four independent experiments. (I) Immunoprecipitation analysis for Coro1B ubiquitination in control or USP45 knockdown HEK293T cells. (J) Quantification of ubiquitin (Ub) levels normalized to Coro1B in I. Data are shown as mean ± SEM of three independent experiments. (K) Immunoprecipitation analysis of Coro1B ubiquitination in cells expressing Flag-Coro1B, Myc-tagged USP45-WT or the catalytically inactive USP45-C199A (C > A) mutant. (L) Quantification of Ub levels normalized to Coro1B in K. Data shown as mean ± SEM of three independent experiments. Significance was determined using one-way ANOVA and Dunnett’s (D, J, and L) or Tukey’s (F) multiple comparisons test, and Student’s t test (H); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS not significant. Source data are available for this figure: SourceData F5.

USP45 interacts with and deubiquitinates Coro1B. (A) Coimmunoprecipitation (Co-IP) analysis of the interaction between Myc-USP45 and Flag-Coro1B or Flag-Coro1C in HEK293T cells. Co-IP experiments were performed using an anti-Myc antibody, followed by a Western blot analysis with the indicated antibodies. (B) (top). Schematic presentation of domain structures and deletion mutants of Coro1B. β-propeller, β-propeller domain; CC, coiled-coil domain. (bottom) Coimmunoprecipitation assays to map the interaction regions between USP45 and Coro1B. (C) Western blot analysis of the expression levels of Coro1B and Coro1C with indicated antibodies in control and USP45 knockdown HeLa cells. (D) Quantification of Coro1B and Coro1C expression normalized to tubulin and GAPDH, respectively. Data are shown as mean ± SEM of at least three independent experiments. (E) Western blot analysis of the expression levels of Coro1B in control (scr) and USP45 knockdown HeLa cells with or without treatment of proteasome inhibitor MG132 (5 μM, 4 h). (F) Quantification of Coro1B expression normalized to GAPDH in E. Data are shown as mean ± SEM of four independent experiments. (G) Cycloheximide (CHX) chase analysis of Coro1B expression in control and USP45 knockdown HeLa cells treated with CHX for the indicated times. (H) Quantification of Coro1B levels normalized to GAPDH at different time points after CHX treatment, as indicated in G. Data shown as mean ± SEM of four independent experiments. (I) Immunoprecipitation analysis for Coro1B ubiquitination in control or USP45 knockdown HEK293T cells. (J) Quantification of ubiquitin (Ub) levels normalized to Coro1B in I. Data are shown as mean ± SEM of three independent experiments. (K) Immunoprecipitation analysis of Coro1B ubiquitination in cells expressing Flag-Coro1B, Myc-tagged USP45-WT or the catalytically inactive USP45-C199A (C > A) mutant. (L) Quantification of Ub levels normalized to Coro1B in K. Data shown as mean ± SEM of three independent experiments. Significance was determined using one-way ANOVA and Dunnett’s (D, J, and L) or Tukey’s (F) multiple comparisons test, and Student’s t test (H); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS not significant. Source data are available for this figure: SourceData F5.

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