Nesd interacts with both centralspindlin components in vivo and in vitro. (A) Colloidal Coomassie blue–stained gels of purifications from Drosophila S2 cells expressing RacGAP50C::PrA or Pav-KLP::PrA. The positions of some proteins identified by MS are indicated on the right. (B) Silver-stained gel of Nesd::PrA purification. Bands corresponding to Pav-KLP and RacGAP50C are indicated. The table shows the relative MS score and number of peptides of the top five MS hits. The bait Nesd is indicated in red. The numbers on the left indicate the sizes in kilodaltons of the molecular mass markers. (C) GST::Nesd purified from bacteria was incubated with either RacGAP50C_1–371 or Pav-KLP_461–887, translated, and radiolabeled in vitro and then pulled down using glutathione beads. The colloidal Coomassie blue (CCB) staining of the protein loading is shown at the bottom. On the right is a schematic summary of the positive (+) and negative (−) results of the GST pull-down assays using different RacGAP50C and Pav-KLP fragments. C1, phorbol ester/diacylglycerol binding; Pbl, Pebble; Ani, Anillin; Kinesin, kinesin catalytic domain; CC, coiled coil; RacGAP, RacGAP50C.