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. 2018 Dec 26;294(8):2651–2664. doi: 10.1074/jbc.RA118.005861

Figure 1.

Figure 1.

TRIAD1 and HHARI are required for cellular DCNL1 monoubiquitylation. A and B, IP of CUL5 complexes from cells expressing GFP–control, GFP–TRIAD1, or catalytically inactive GFP–TRIAD (C310S) and detection of co-precipitated proteins by immunoblot analysis as indicated. Nonspecific IgG was used as negative control. *, nonspecific band. C, subfractionation of HEK293 cell extracts (Total) into cytosolic high-salt nuclear extract (NEX), and soluble chromatin (CHEX) fractions. Detection of endogenous DCNL1 and CRLs was done by immunoblot as indicated. Histone H2A served as a chromatin marker. D, HA-IP of HEK293 cells expressing HA–DCNL1 or HA-CUL5 were treated with either ubiquitin-deconjugating enzyme USP2 or the deneddylating enzyme NEDP1 followed by anti-HA immunoblot analysis. E, immunoblot analysis of endogenous DCNL1-Ub in cytosolic fractions of cells expressing WT TRIAD1 (WT), catalytically inactive variants TRIAD1 (C310S), TRIAD1 (C310S, R371A, E382A, and E455A), and constitutively ligase-active variant TRIAD1 (R371A, E382A, and E455A). F, immunoblot analysis of endogenous DCNL1-Ub in cytosolic fractions of cells expressing WT HHARI (WT), catalytically inactive HHARI (C357S) or HHARI (C357S, F430A, E431A, and E503A), and constitutive ligase-active HHARI (F430A, E431A, and E503A).