Fig. 3.

Daf1/Dpb3 and Dpb4 form a heterodimer. (A) Cell lysates from cells expressing Dpb4–TAP and Daf1–GFP and from control cells expressing Daf1–GFP only were subjected to immunoprecipitation with an antibody specific for TAP. Precipitated proteins were analyzed by immunoblotting (IB), using the indicated antibody. (B) Cell lysates from cells expressing Cdc20–FLAG and Daf1–GFP were immunoprecipitated with a FLAG antibody and analyzed by immunoblotting using a GFP antibody. Cells expressing Daf1–GFP only were used as a control. (C) Dpb4 and Daf1 form a stable complex in vitro, as evidenced by the elution profile of gel-filtration chromatography (Right). The purified Dpb4-Daf1 complex was analyzed on SDS–PAGE (Left). C, Dpb4–Daf1 complex; M, molecular weight. (D) GST pull-down assays demonstrated physical binding between Daf1 and Dpb4. Both input and pull-down samples were subjected to immunoblotting with anti-His and anti-GST antibodies.