|
Fig. 11
Sharacterization of interactions between Xirp1 and Filamin C or Enah/Vasp family members.
(A) Table summarizing the results of direct yeast two hybrid experiments with the C-terminus of zebrafish Xirp1 (residues 2097–2297) and the GST-tagged domains 19–21 of zebrafish Filamin Ca (FlnCa) and b (FlnCb). The Filamin CbΔ (FlnCbΔ) isoform represents a splice variant lacking the unique insertion within domain 20 that is present in all Filamin C proteins. (B) Western blot overlay experiments show specific binding of the GST-tagged domains 19–21 of FlnCa and FlnCb to the blotted C-terminus of Xirp1. GST and FlnCbΔ do not bind, confirming that the unique insertion within domain 20 of Filamin Cb is necessary for the interaction. (C) Co-immunoprecipitation experiments confirm these interactions: FlnCa and FlnCb are co-precipitated, whereas GST alone is not. (D) Western blot overlay experiments show specific binding of T7-tagged N-terminus of zebrafish Xirp1 (residues 1–58) containing proline-rich repeat1 (PR1) to the blotted EEF-tagged EVH1 domains of zebrafish Enah, VaspI and VaspII. (E) Summary of binding assays shows the isoforms of Xirp1 identified in our study, and the respective binding sites for EVH1 domains of Enah and Vasp (PR1) and Filamin C (carboxy-terminus). Binding to F-actin is presumably mediated by Xin-repeats (red boxes). PRs are depicted by blue boxes.