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. 2019 Dec 1;33(23-24):1702–1717. doi: 10.1101/gad.332395.119

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© 2019 Wu et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Figure 5. — Cezanne promotes Rap80/Abraxas/BRCA1 recruitment through its K11-linkage DUB activity. (A,B) Depletion of Ube2S in Cezanne knockdown cell rescues Rap80 IRIF. Cells were treated with 10 Gy IR, followed by 2 h incubation before fixation and staining. Representative images (A) and quantification of Rap80, Abraxas, and BRCA1 IRIF (B) are shown. Nonparametric Kruskal–Wallis ANOVA was used for statistical analysis. (C) Double depletion of Ube2S and Cezanne rescues the repair deficiency of Cezanne-depleted cells. U2OS cells treated with indicated siRNAs were treated with 2Gy IR, fixed, and stained with γ-H2AX antibody at indicated times. The percentage of γ-H2AX foci positive cells were quantified. Nonparametric Kruskal–Wallis ANOVA was used for statistical analysis. (D) Depletion of Ube2S rescues the IR sensitivity of Cezanne KO cells detected by colony formation assay. Student's t-test was performed to compare Cez KO treated with siCon (orange) or siUbe2S (green) at indicated IR doses. “Control” (black) represents cells established from transfection with the construct without sgRNA. (E) Overexpression of GFP-tagged Ube2S in U2OS cells decreases Rap80/Abraxas/BRCA1 IRIF. Student's t-test was used for statistical analysis. (F) In vitro GST-pull-down assay using purified recombinant GST-Cezanne UBA or Rap80-UIMs incubated with K63-linked tetra-ubiquitin (K63-Ub4), tri-ubiquitin (K63-Ub3), mixed K63/K11-linkage tri-ubiquitin (K63/K11-L), or branched K63/K11- tri-ubiquitin (K63/K11-B).