Fig. 5. Immunoprecipitation of Echinoid reveals homophilic interactions and interactions with Egfr. (A) Cultured S2 cells were transfected with EdFLAG and Edmyc to evaluate homophilic interactions. Immunoprecipitation with anti-FLAG Sepharose co-precipitates Edmyc; co-precipitation is not significantly affected by transfection with Egfr. (B) Analysis of immunoprecipitated Echinoid-FLAG (EdFLAG) revealed that a clipped form of Echinoid is also present, corresponding approximately to the 500 C-terminal amino acids (arrow, left blot). The amount of the clipped form is not altered by Egfr transfection. An antibody against the N-terminal region of Echinoid (Bai et al., 2001) detects a smaller form of Echinoid in media from these cells (right blot); this may represent shedding of the immunoglobulin and fibronectin III domains of Echinoid into the extracellular space. (C) Echinoid associates with Egfr. (Left panel) Cultured S2 cells were transfected with 1.8 µg Egfr DNA and variable amounts of EdFLAG DNA. Immunoprecipitation of EdFLAG co-precipitated increasing amounts of Egfr. (Middle and right panels) Cultured S2 cells were transfected with Egfr and EdFLAG or Edmyc DNA. Immunoprecipitation with FLAG-Sepharose or myc-Sepharose co-precipitated Egfr. Transfection of Egfr and other FLAG and myc-tagged proteins (morgueFLAG, erkmyc) did not lead to co-precipitation of Egfr. (D) Immunoprecipitation of Egfr from cultured S2 cells transfected as in (C) co-precipitated EdFLAG. (E) Echinoid constructs used in these experiments: full-length Echinoid contains seven Ig repeats and a FN3 repeat on its extracellular face. EdFLAGN lacks the Ig repeats and the N-terminal part of the FN3 domain (amino acids 69-787) but not the signal sequence. EdFLAGC lacks the tyrosine-rich intracellular domain (amino acids 1078-1332).