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Figure 2


Fig. 2. Ubiquitylation of Dock180 in HEK293T cells. (A) Results of an in vivo ubiquitylation assay. HEK293T cells were transfected with the indicated plasmids, and after treatment of 10 µM MG-132 for 12 hours, cells were lysed and subjected to immunoprecipitation (IP) and immunoblotting (IB). MIG, normal mouse immunoglobulin; TCL, total cell lysate; Ub, ubiquitin. (B) For the urea-reversal immunoprecipitation, the initial precipitates from the anti-Dock180 mAb were treated with 8 M urea buffer for 1 minute and then immunoprecipitated again using the same antibody. (C) MCAS and HEK293T cells were treated with 20 µg/ml cyclohexamide and 10 µg/ml MG-132 for 24 hours as indicated at the top of the panel. Cell lysates were immunoblotted with anti-Dock180 antibody to detect endogenous Dock180.





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