Figure 3.

Wdr8 physically interacts with Pkl1/Kinesin-14 through Msd1. (A) Msd1 and Pkl1 interact in the presence or absence of Wdr8. Pull-down assays were performed by using the GFP-trap system. Extracts were prepared from cells containing Msd1-GFP alone (lanes 1 and 5), Pkl1-13myc alone (lanes 2 and 6), or both in wdr8⁺ (lanes 3 and 7) and wdr8Δ mutant (lanes 4 and 8). Immunoblotting with anti-myc (top) or anti-GFP antibodies (bottom) against whole cell extracts (WCE; lanes 1–4) and pull-down precipitates (lanes 5–8) is shown. (B) Wdr8 interacts with Pkl1 only in the presence of Msd1. Pull-down was performed as in A by using extracts prepared from cells containing Pkl1-GFP alone (lanes 1 and 5), Wdr8-13myc alone (lanes 2 and 6), or both in msd1⁺ (lanes 3 and 7) and msd1Δ mutant (lanes 4 and 8). (C) Msd1 and Wdr8 interact in the presence or absence of Pkl1. Pull-down was performed as in A by using extracts prepared from cells containing Msd1-GFP alone (lanes 1 and 5), Wdr8-13myc alone (lanes 2 and 6), or both in pkl1⁺ (lanes 3 and 7) or pkl1Δ mutant (lanes 4 and 8). In A–C, asterisks denote unspecific bands. (D) Summary of physical interactions among the three proteins. Msd1 interacts with Pkl1 or Wdr8 in the absence of Wdr8 or Pkl1, respectively (solid lines). Wdr8 binds to Pkl1 only in the presence of Msd1 (dotted line). WT, wild type.