Panel A shows SDS-PAGE images and a schematic summarizing tubulin-binding activities of multiple Ana1 protein fragments spanning different structural regions. Panel B shows multiple sequence alignments highlighting conserved Ana1 amino acid residues and mutation sites across several Drosophila species. Panel C shows isothermal titration calorimetry thermograms and binding isotherms measuring interactions between Ana1 fragments and the Cep135 protein domain. Panel D shows immunoblots comparing binding of GFP-Ana1 wild-type and mutant proteins with Flag-Cep135 in bound and input protein fractions.
Mapping of the Ana1-MT interaction region. (A) SDS-PAGE analysis showing the region of Ana1 that directly interacts with MTs. A schematic representation of the various Ana1 constructs analyzed for binding is shown. (B) MSA showing the conservation of Ana1 residues involved in MT binding in Drosophila species. (C) ITC experiments showing interactions between Cep135257–351 and WT Ana1 or MT binding–deficient mutant (Ana1RKN). Mutations within the Ana1-MT–binding interface do not affect its interaction with Cep135. (D) S2 cells were transiently cotransfected with GFP-tagged Ana1 and Flag-tagged Cep135, along with various MT-binding mutants of Ana1 (RKNC, RKN, and RKC). Cell extracts were subjected to pull-down assays using GFP-Trap beads, and bound proteins were analyzed by western blotting with anti-Flag to detect Cep135. All three Ana1 mutants (RKNC, RKN, and RKC) retained the ability to bind Cep135. The experiment was repeated three times with similar results. WT, wild type; MSA, multiple sequence alignment. Source data are available for this figure: SourceData FS4.