Figure 3.

Stabilization of the activated form of Blk by the proteasome inhibitor (MG132) and detection of multiubiquitinated forms of Blk. (A) Western blot of Cos-7 cells transfected with Blk with or without pervanadate (100 μM) and MG132 (25 μM). Total cell lysates were probed with anti-Blk antibody and were subjected to immunoprecipitation with the same antibody and immune-complex kinase assay. (B) Blk becomes ubiquitinated on pervanadate treatment. Cos-7 cells transfected with Blk were treated with or without pervanadate (100 μM) and MG132 (25 μM) for 2 hr. Total cell lysates (1.5 mg) were immunoprecipitated with anti-Blk antibody, and ubiquitinated Blk was detected by Western blot analysis with antiubiquitin antibody. (C) Degradation of endogenous Blk by kinase activation and stabilization with MG132 treatment. The LS102.9 cells (2 × 10⁷) were incubated in the absence or presence of antibody against murine IgM (30 μg/ml) with or without MG132 (40 μM) as indicated. The lysates were immunoprecipitated with anti-Blk antibody conjugated with protein A agarose beads, then were subjected to immunoblotting for Blk.