NSC697923 specifically inhibits Ubc13-mediated polyubiquitin chain synthesis in vitro. (A) NSC697923 inhibits the synthesis of polyubiquitin chains catalyzed by Ubc13-Uev1A in vitro. The in vitro reaction, which contains purified E1, Ub, Ubc13, and Uev1A, was carried out with or without the indicated concentrations of NSC697923. The reaction products were analyzed by an anti-Ub Ab on Western blots. The asterisk (*) denotes the signal from the cross-reaction of the Ab with the recombinant Ubc13 protein. (B) Same as in panel A, except that purified GST-TRAF6 was included as the E3 in the complete reaction as indicated. (C) NSC697923 exhibits no inhibitory effect on UbcH5c-mediated polyubiquitin chain synthesis. The assay was carried out as in panel B, except that UbcH5c instead of Ubc13-Uev1A was used as the E2 in the ubiquitination reaction. (D) NSC697923 has no inhibitory effect on the formation of Ubc13-Uev1A complex in vitro. Purified GST or GST-Ubc13 protein was incubated with the cell extracts prepared from OCI-Ly10 cells with or without the presence of NSC697923 as indicated. The amounts of Uev1A protein associated with GST or GST-Ubc13 were analyzed by Western blotting. The first lane was loaded with 8% of the input cell lysate. (E) NSC697923 inhibits the formation of the Ubc13∼Ub thioester conjugate. The assay was carried out as described in panel A, except that the loading buffer for SDS-PAGE gel contained no reducing agents unless otherwise specified and the reaction was analyzed by Western blotting with a Ubc13-specific Ab. (F) Formation of the UbcH5c∼Ub conjugate is not inhibited by NSC697923. The assay was carried out as in panel E, except that UbcH5c instead of Ubc13 and Uev1A was used in the reaction and a UbcH5c-specific Ab was used for Western blot analysis.