Fig. 5

Fig. 5

Fig. 5. zUba7 initiates ubiquitination cascades. (A) zUba7 activates Myc-Ub. The activation was analyzed by immunoblotting with anti-Myc. (B) Ub activation by zUba7 requires ATP and Cys 590, and it is sensitive to DTT. (C) Pull down of Flag-Strep-zUba7 (FT-zUba7) and HA-Ub. FT-zUba7 and HA-Ub were expressed in HEK293T cells either alone or together, as indicated. After pull down against the strep tag of zUba7, samples were separated by SDS-PAGE under non-reducing or reducing conditions and were immunoblotted with anti-HA and anti-FLAG antibodies. (D) zUba7 and zUba1 have distinct E2 charging activities in vitro. Assays employed E2s produced in Escherichia coli extracts, HA-Ub and the indicated E1, as described in the Methods. (E) Bacteria expressing Myc-zMdm2 were incubated in the absence or presence of zUba1, zUba7, Ub, and zUbcH5b as indicated. After 1 h at 30 °C, samples were analyzed by immunoblotting with the indicated antibody. (F) In vitro ubiquitination of Flag-zP53 by Myc-zMdm2. (G) Immunoblotting reveals that zUba7 overexpression rescues the Ub level of Zuba1^−/−.