Depletion of USP45 causes enrichment of V-ATPase at the lysosome. (A) Immunofluorescent analysis of the colocalization of transiently transfected Myc-USP45 and endogenous vesicle markers (EEA1, Rab7, LAMP1, and LC3) in HeLa cells using the indicated antibodies. Scale bar, 10 μm (original) and 4 μm (zoom-in). (B) Quantification of colocalization of Myc-USP45 and markers in A. Data are shown as mean ± SEM in n = 3, ≥15 cells from three independent experiments. (C) RT-PCR analysis of USP45 expression in control and USP45 knockdown HeLa cells. (D) Immunofluorescent analysis of LysoTracker (LTK) and lysosome (LAMP2) in USP45 knockdown cells. Scale bar, 10 μm. (E) Quantification of LTK dot number and LAMP2 intensity respectively in D. Data shown as mean ± SEM in n = 3, ≥40 cells. (F) Immunofluorescent analysis of colocalization of V-ATPase subunit (V1H) and lysosome (LAMP1). Scale bar, 20 μm. (G) Quantification of colocalization of V1H and LAMP1 in F. ± SEM in n = 3, ≥20 cells. (H) Lysosomal immunoprecipitation (LysoIP) analysis of V-ATPase subunits (V1Band V0D) in enriched lysosome lysate by precipitating TMEM192-HA (TMEM192-Flag as negative control) in control and USP45 knockdown HEK293T cells. V-ATPase subunits (V1B, V0D) and organelle markers (lysosome, LAMP2; mitochondria, ATP5A; Golgi, GM130; and ER, Calnexin) were detected by antibodies. (I) Quantification of V1B normalized to HA. Data are shown as mean ± SEM from three independent experiments. Significance was determined using one-way ANOVA and Tukey‘s (B) or Dunnett’s (E and I) multiple comparisons test, and Student’s t test (G); *P < 0.05; ***P < 0.001; ****P < 0.0001. Data shown as mean Source data are available for this figure: SourceData FS2.