Figure 4.

Mutant S294A PR-Bs fail to undergo ubiquitination. HeLa cells were transiently cotransfected with expression vectors encoding HA-tagged-ubiquitin and either epitope-flag-tagged wild-type PR-B [wt-PR-B:flag (lanes 1–3)] or epitope-flag-tagged S294A mutant PR-B [S294A:flag (lanes 4–6)] and treated for 4 h without (lanes 2 and 5) or with R5020 (lanes 3 and 6). PRs were immunoprecipitated by using an anti-flag M2 affinity gel and were visualized by immunoblotting with either HA- or flag-specific antibodies. Lanes 1 and 3, nonspecific antibody and similar affinity gel. High-molecular-weight ubiquitinated forms in immunoprecipitates from lysates of cells containing wild-type but not S294A mutant PR-Bs are indicated by arrows.