Figure 2.

Cbl promotes ubiquitylation of CrkL. (A) 293T cells were transfected with plasmids as indicated, and the cells were left untreated or were treated with pervanadate (PV) for 30 min. The cell lysates were immunoprecipitated (IP) with anti-CrkL, blotting was carried out and detection was performed using anti-haemagglutinin (anti-HA). Poly-ubiquitylated CrkL ((Ub)_n–CrkL) is indicated. The same membrane was reprobed with anti-CrkL. 'H' indicates the position of the IgG heavy chain. (B) Cells that coexpress wild-type Cbl or the Cbl RING-finger mutants Cbl-CA or Cbl-WA were analysed as in (A). (C) Thymocytes from Cbl^+/+ and Cbl^−/− mice were either unstimulated or were stimulated with anti-CD3 for the durations indicated, followed by immunoprecipitation with anti-CrkL, blotting, and detection using anti-ubiquitin (anti-Ub).