Cbl ubiquitinates p85 in vitro. (A) T7-tagged p85 immunoprecipitated from HEK293T cells using anti-T7 antibody and Protein A beads was incubated with 200 ng of recombinant Cbl purified from BL21 in in vitro ubiquitination assay using recombinant E1, E2 (UbcH5B), and Flag-tagged wild-type Ub. After extensive washing, ubiquitinated p85 species were eluted from Protein A beads by SDS sample buffer and immunoblotted with the indicated antibodies. (B) p85 can be ubiquitinated at multiple sites. An in vitro ubiquitination assay was performed with a lysine-less ubiquitin mutant (KR) that cannot form ubiquitin chains. (C) p85 ubiquitination is lost in Cbl^−/− MEFs and is restored in Cbl^−/− MEFs reconstituted with Cbl.