FIG. 4.
UBE2E3 participates in the Nedd4-2-dependent ubiquitination cascade. (A) In vitro-translated 35S-labeled Nedd4-2 was incubated (+) for 1 h at 30°C with ubiquitin, E1, and different E2s. Samples were then analyzed by SDS-PAGE and autoradiography. The position of autoubiquitinated Nedd4-2 species (Nedd4-2-Ub) in lane 5 are indicated by brackets to the right of the gel. (B) Equal amounts of crude lysate from E. coli were incubated (+) for 2 h at 30°C with ubiquitin, E1, wild-type UBE2E3, or UBE2E3-CS and Nedd4-2 and then analyzed by SDS-PAGE and Western blotting using a monoclonal antibody directed against ubiquitin (α-ubiquitin). IB, immunoblotting.