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. Author manuscript; available in PMC: 2011 Dec 15.
Published in final edited form as: Immunity. 2010 Dec 14;33(6):863–877. doi: 10.1016/j.immuni.2010.11.027

Figure 7. RTA mediates RAUL deubiquitination through Usp7.

Figure 7

(A): Endogenous RAUL and Usp7 interact. BCBL1 cells were treated with TPA for 24 hr and cell lysates were immunoprecipitated with anti-Usp7 followed by RAUL immunoblot to detect bound protein. (B and C): Usp7 deubiquitinates RAUL in vivo and in vitro. 293T cells were transfected with Myc-RAUL, WT Usp7, Usp7-cs and HA-Ub. The cells were treated with MG132 for 6 hr before harvest. Polyubiquitinated forms of RAUL were detected by immunoprecipitation with anti-Myc followed by HA immunoblot. (C): Purified RAUL protein (400 nM) plus E1, E2 (UbcH5a) and Ub were incubated at 37°C for 1 hr to catalyze self-ubiquitination. The Ub-RAUL protein was then purified by immunoprecipitation with anti-RAUL antibody and incubated with purified Usp7 protein or its catalytically inactive mutant (Usp7-cs) in deubiquitination buffer at 37°C for 2 hr. The resulting reactions were subjected to immunoblot with RAUL. (D): Usp7 upregulates RAUL protein amounts. Colon cancer cell lines HCT116 Usp7+/+ or Usp7−/− cells were immunblotted with the indicated antibodies to evaluate both RAUL and its target substrate IRF3. (E): Usp7 enhances the E3 activity of RAUL toward IRF3 in vitro. Immunoprecipitated purified RAUL protein from either RAUL expressing 293T cells or RAUL+Usp7 expressing 293T cells were incubated with its substrate GST-IRF3, plus E1, UbcH5a and Ub in vitro at 37°C for 1 hr. Conjugated IRF3 was determined by Ub immunoblot. (F): RTA increases the recruitment of Usp7 to RAUL complexes. TRExBCBL1 cells were treated with Doxycyclin (Dox) for 12 hr to activate RTA expression. Endogenous RTA-Usp7-RAUL interaction was evaluated by immunoblot with the indicated antibodies. (G):RTA promotes RAUL deubiquitination. HCT116 (Usp7+/+ or Usp7−/−) cells were electroporated with the indicated amounts of RTA plasmid and RAUL self-ubiquitition was determined by Ub immunoblotting. Unconjugated RAUL and RTA were measured by RAUL or RTA immunoblotting. (H): RTA enhances the ability of RAUL to degrade IRF7 via Usp7. 293T cells were cotransfected with IRF7, Myc-RAUL, RTA and shRNA-Usp7 as indicated, and IRF7 protein amounts were evaluated by IRF7 immunoblotting. (I): RTA and RAUL cooperate in suppressing IRF3-mediated IFNβ reporter activity. 293-TLR3 cells were transfected with the indicated combinations of IRF3, RAUL, RTA, shRNA- Usp7 and IFNβ-LUC plasmids and the cells were treated with poly I:C for 9 hr to mimic TLR3 activation. IRF3-mediated IFN-β reporter activity in the cells that were pretreated shRNA-RAUL or scrambled IFN-β activation was determined by luciferase assays.