Fig 1: The USP12/46-WDRs complex deubiquitinates Itgb1 on endosomes.(A, B) In vitro deubiquitination assay using recombinant WDR48-WDR20-USP12 (WT or C48S) complex, UCHL5 or USP7, and ubiquitinated Itgb1 enriched from USP12/46-dKOESCRT-KD fibroblast cell lysate followed by WB (A) for ubiquitin with quantification (B) of Itgb1 ubiquitination levels. The intensity of the Ub signals was normalized to the intensity of the IP-ed Itgb1 signals. Statistical analysis was carried out by RM two-way ANOVA with Dunnett’s multiple comparison test comparing the BSA group with USP12WT, USP12C48S, UCHL5, or USP7 group in ESCRT-KD condition (P = 0.0194, 0.2707, 0.9997, and 0.9844, respectively). *P < 0.05; n.s. not significant. Data were shown as Mean ± SD, n = 3 independent experiments. BSA served as a negative control. (C) Coomassie-blue staining of recombinant proteins used in the in vitro deubiquitination assay. MS was used to determine the identity of the protein bands indicated in the DUB complexes. Band *1, WDR48; band *2, WDR20; band *3, a cleaved WDR20; band *4, USP12. (D) Representative structured illumination microscopy (SIM) images of Itgb1 and Ub in USP12/46-dKO fibroblasts stably expressing EGFP-USP12 treated with or without ESCRT-KD siRNAs. Boxes indicate magnified cell regions displayed in the Zoom panel. White/pink arrowheads show the Itgb1-labeled FAs, and white arrows show the direction of line profiles. Scale bar, 10 µm. (E–G) Superplots showing the PCC between Ub and EGFP-USP12 (E), Ub and Itgb1 (F), and Itgb1 and EGFP-USP12 (G) in USP12/46-dKO fibroblasts stably expressing EGFP-USP12 and treated with or without ESCRT-KD siRNAs. Statistical analysis was carried out by two-sided Welch’s t-test. The P values in (E–G) are 0.0287, 0.0011, and 0.0048, respectively. *P < 0.05; **P < 0.01. Data were shown as Mean ± SD, n = 3 independent experiments, in total 42 cells per condition. (H) Representative SIM images of Itgb1 and EEA1 in USP12/46-dKO fibroblasts stably expressing EGFP-USP12 treated with or without ESCRT-KD siRNAs. Boxes indicate magnified cell regions displayed in the Zoom panel. White/pink arrowheads show the Itgb1-labeled FAs, and white arrows show the direction of line profiles. Scale bar, 10 µm. (I–K) Superplots showing the PCC between EEA1 and EGFP-USP12 (I), EEA1 and Itgb1 (J), and Itgb1 and EGFP-USP12 (K) in USP12/46-dKO fibroblasts stably expressing EGFP-USP12 and treated with or without ESCRT-KD siRNAs. Statistical analysis was carried out by two-sided Welch’s t-test. The P values in (I–K) are 0.0178, 0.0040, and 0.0208, respectively. *P < 0.05; **P < 0.01. Data were shown as Mean ± SD, n = 3 independent experiments, in total 42 cells per condition. Source data are available online for this figure.