Spindly^SPDL-1 binds Zwilch^ZWL-1 and the ROD-1 β-propeller. (A) Lysates of insect Sf21 cells coinfected with viruses to express full-length (FL) ROD-1^1–2,177ZZ, ROD-1^1–1,203ZZ, ROD-1^1–372–Zwilch^ZWL-1, or Zwilch^ZWL-1 alone. ROD-1 and Zwilch^ZWL-1 were tagged with 6×His for detection on immunoblots. (B) Coomassie-stained protein gels showing purified recombinant GST::Spindly^SPDL-1 used in pull-downs. (C and D) GST pull-downs from the lysates in A with purified GST::Spindly^SPDL-1 from B. Protein fractions bound to beads were analyzed by immunoblotting using anti-6×His antibody. The same membranes were then reprobed with anti-GST antibody. (E) Lysates of insect Sf21 cells infected with viruses encoding for the ROD-1 β-propeller (residues 1–372) or Zwilch^ZWL-1, both tagged with 6×His. (F) Immunoblots of GST pull-downs from the lysates in E with purified GST::Spindly^SPDL-1 (see A). (G) Coomassie-stained protein gels showing purified ROD-1 β-propeller alone and in complex with wild-type (WT) Zwilch^ZWL-1 or the E/A mutant. (H and I) Immunoblots of GST pull-downs using the purified proteins in B and G. (J) Cartoon showing the bipartite interaction between RZZ and the C-terminal domain (CTD) of Spindly^SPDL-1. Molecular mass is indicated in kilodaltons.