Fig. 1. Interaction between Citron proteins and TTC3. (A) Domain structure of Citron proteins, TTC3 and the described mutants. Kinase domain (black), Rho-binding domain (black-box), coiled coil region (grey), C1 domain (cyan), PH domain (pink), CNH domain (red), Proline-rich region (orange), PDZ-binding domain (brown), TPR motifs (yellow), Citron binding region CBR (green) and ring finger domain (blue) are indicated. The sequences used as bait, and found as prey are indicated. (B) Total cell lysates from HEK293 cells co-transfected with GFP-CIT-N and Myc-CBR were immunoprecipitated with anti-Citron antibodies and analyzed by western blotting as indicated. (C) Total cell lysates from HEK293 cells co-transfected with GFP-TTC3 and CIT-N or CIT-K were immunoprecipitated with anti-Citron antibodies and analyzed by western blotting as indicated. (D) PC12 cells were co-transfected with GFP-TTC3 and allowed to differentiate by NGF treatment for 2 days after transfection. Total cell lysates were then immunoprecipitated with anti-GFP antibodies and endogenous CIT-K was revealed by western blotting. (E) The TTC3 mutants Cterm and CBR were co-transfected with CIT-K and analyzed as in C. (F) Myc-CIT-K was co-transfected in HEK293 cells with GFP-TTC3 (upper panel) or with GFP-CBR (lower panel). Bar, 10 µm.