Panel A: A schematic representation of various Ana1 and Cep135 deletion constructs. The diagrams show the regions of Cep135 and Ana1 that bind to each other. Panel B: Western blot images showing the results of pull-down experiments with S2 cells co-transfected with GFP-tagged Ana1 and Flag-tagged Cep135 fragments. The blots are labeled with molecular weight markers and show bound and input fractions. Panel C: Western blot images showing the results of pull-down experiments with S2 cells co-transfected with GFP-tagged Cep135-N and Flag-tagged Ana1 fragments. The blots are labeled with molecular weight markers and show bound and input fractions. Panel D: Isothermal titration calorimetry (ITC) graphs showing direct interaction between Cep135 and Ana1. The graphs display power (DP) versus time and enthalpy change (delta H) versus molar ratio, with binding constants indicated. Panel E: Western blot images showing the results of pull-down experiments with S2 cells co-transfected with GFP-tagged Cep135 and Flag-tagged Ana1 with deletions in the binding regions. The blots are labeled with molecular weight markers and show bound and input fractions.
Ana1 binds to Cep135 via two distinct interfaces. (A) Schematic representation of the various Ana1 or Cep135 deletion constructs analyzed in this study. The N-terminal half of Cep135 (aa1–498, referred to as Cep135-N) binds to Ana1, whereas the C-terminal half (aa498–1059, referred to as Cep135-C) does not, while for Ana1, the N-terminal fragments aa1–180 and aa180–497 both bind to Cep135-N, but the C-terminal half does not. (B) S2 cells were transiently cotransfected with GFP-tagged Ana1 and Flag-tagged Cep135 fragments. Extracts were then subjected to pull-down experiments using GFP beads, and bound fractions were analyzed by western blotting. The experiment was repeated three times with similar results. (C) S2 cells were transiently cotransfected with GFP-tagged Cep135-N and Flag-tagged Ana1 fragments. Extracts were then subjected to pull-down experiments using GFP beads, and bound fractions were analyzed by western blotting. The experiment was repeated three times with similar results. (D) ITC-based experiments showing direct interaction between Cep135 and Ana1. 400 μM Cep135 fragments in the syringe were titrated into 40 μM Ana1 fragments in the cell. The experiment was repeated three times, and the average Kd value is displayed. (E) S2 cells were transiently cotransfected with GFP-tagged Cep135 and Flag-tagged Ana1, which carries deletion of the two identified binding regions. Extracts were then subjected to pull-down assays using GFP beads and were analyzed by western blotting. The experiment was repeated three times with similar results. Source data are available for this figure: SourceData F3.