FIGURE 5.

Enhancement of Rta ubiquitination by RNF4 in vitro. A, conjugation of ubiquitin to Rta was analyzed in vitro using purified Rta, an Rta mixture that contains SUMO-2-conjugated Rta, and GST-RNF4 in the presence of ubiquitin (Ub), ubiquitin E1-activating enzyme, and ubiquitin E2 enzyme (UbcH5a). Ubiquitinated proteins were examined by immunoblotting (IB) using anti-Rta antibody. B, similar in vitro Ub assay was conducted using FLAG-ubiquitin (FLAG-Ub), purified Rta, SUMO-2-conjugated Rta, E1, E2, and GST-RNF4. Thereafter, Ub-Rta was immunoprecipitated (IP) using anti-FLAG antibody and then detected by immunoblotting using anti-Rta antibody. C, additionally, the ubiquitination of RNF4 was examined in vitro with purified GST-RNF4, Rta, or SUMO-2-conjugated Rta in the reaction mixture containing ubiquitin (Ub), ubiquitin E1-activating enzyme, UbcH5a, or ATP. Ubiquitinated proteins including monoubiquitinated, diubiquitinated, and polyubiquitinated GST-RNF4 were detected by immunoblot analysis using anti-GST antibody. Asterisks indicate bands detected nonspecifically.