FIGURE 8.

CopA3 directly enhances ubiquitin ligase activity. A, in vitro ubiquitination assays were carried out with the S5a substrate, the E1, E2, and RING E3 ligases, and ubiquitin in the presence or absence of CopA3 (20 μg/ml) or P4 (20 μg/ml). Reactions were initiated by the addition of ATP and samples were incubated at 37 °C for 1 h. The presented results are representative of three independent experiments. B, HT29 cell lysates were prepared and immunoprecipitated with an anti-p21^Cip1/Waf1 antibody. C, immunoprecipitated p21^Cip1/Waf1 was added to the ubiquitination assay reaction buffer in place of S5a, and ubiquitinated p21^Cip1/Waf1 was visualized with antibodies against p21^Cip1/Waf1 or ubiquitin. IB, immunoblot; IP, immunoprecipitation.