CC fragments interact with the dynein complex. (A) Dynein pull-down assays. Yeast extracts expressing Pac11-13Myc or Dyn1_TAIL-3GFP were incubated with GST fusion proteins (top left) or S-tagged proteins (top right) immobilized on glutathione beads or S-protein agarose, respectively. Bound proteins were eluted and analyzed by immunoblotting (middle and bottom). CB, Coomassie blue; WT, wild type. (B) In vivo interaction of CC^1–303 with Dyn1 in the BiFC assay. The N- and C-terminal fragment of Venus, VN and VC, respectively, were fused to the C terminus of Dyn1 and the N terminus of Num1 or CC^1–303. Fluorescence as a result of reconstitution of the Venus fluorophore was acquired in the YFP channel. Arrows indicate Venus foci at the SPB. Bars, 1 µm. (C, left) The percentage of binucleated cells in cultures grown in YP media containing 2% raffinose and 2% galactose at 12°C for 16 h. Error bars are standard error of proportion (n > 600 cells for each strain). (right) A Western blot of total cell lysates prepared from cultures indicated on the left. Pgk1 was used as a loading control. The CC^1–325-13Myc level induced by the GAL1 promoter (lane 2) was 2.4-fold higher than that expressed by the endogenous NUM1 promoter (lane 4).