FIG. 7.

Cdc34 self-association is facilitated by Ub thiolester formation. (A) Presence of Ub in Cdc34 coimmunoprecipitates. Myc- and Flag-tagged Cdc34 were coexpressed together with HA-tagged Ub in YPH499 cells. Myc-Cdc34 was immunoprecipitated (IP) from cell extracts by using an anti-Myc antibody. The Myc-Cdc34, Flag-Cdc34, and HA-Ub contained in the immunoprecipitate were separated by SDS-PAGE and detected by immunoblotting (IB) with anti-Myc (left panel), anti-Flag (middle panel), and anti-HA (left panel) antibodies. The replacement of Cdc34 by Cdc34 C95A served as the negative control. The asterisk indicates a proteolytic product of Cdc34. Protein expression levels within the lysates used for immunoprecipitations are shown at the bottoms of the respective panels. wt, wild type. (B) Dependence of Cdc34 self-association on thiolester formation. The procedure was the same as for panel A except that extracts were treated with or without DTT followed by dialysis to remove DTT prior to immunoprecipitation.