Fig. 1. NRAGE interacts with Che-1. (A) Schematic representation of Che-1 C-terminal deletion mutants and full-length proteins. Construct names are indicated on the left. (B) Yeast two-hybrid assay. AH109 yeast cells were co-transformed with the indicated constructs and plated onto SD media lacking leucine and tryptophan (–LW) to verify the expression of both bait (W⁺) and prey (L⁺) plasmids, or onto media lacking leucine, tryptophan, histidine and adenine (–LWHA) to examine the interaction between bait and prey proteins. (C) Western blot analysis of lysates from NIH3T3 cells, transfected with expression constructs for Myc-NRAGE or vector expressing Myc-tag only, immunoprecipitated (IP) with anti-Myc monoclonal antibody. Anti-Che-1 polyclonal antibody was used to co-immunoprecipitate endogenous Che-1 protein in Myc-NRAGE expressing cells. (D) CGNs obtained from day 8 postnatal (P8) rats cultured in vitro for 6 days were processed for co-immunoprecipitation assay. Immunoblotting with anti-NRAGE polyclonal antibody showed endogenous NRAGE protein in samples immunoprecipitated with anti-Che-1 rabbit serum (I.P.-Che-1). No immunoreactivity from samples immunoprecipitated with normal rabbit serum (I.P.-control) was detected. (E) Dual-label fluorescence microscopy was performed in fixed CGNs to obtain immunolocalization of endogenous NRAGE (green) and Che-1 (red), as described in Material and Methods. Extensive colocalization (yellow) between NRAGE and Che-1 is visualised by the merged-colour image.