Figure 2

The interaction between ECRG4 and ECRG1 was verified by co-immunoprecipitation assay in vivo. (A) Detection of ECRG4 and ECRG1 protein expression in transfected cells (pcDNA3.1, pcDNA3.1-His-ECRG4, pcDNA3.1-FLAG-ECRG1 and pcDNA3.1-His-ECRG4+FLAG-ECRG1) by Western blot. FLAG-ECRG1 or His-ECRG4 protein was detectable in EC9706/pcDNA3.1-FLAG-ECRG1 or EC9706/pcDNA3.1-His-ECRG4 cells, respectively, and both in EC9706/pcDNA3.1-His-ECRG4+pcDNA3.1-FLAG-ECRG1 cells. (B) EC9706 cells, transiently transfected with FLAG-ECRG1 and His-ECRG4, were immunoprecipitated by anti-FLAG antibody and detected by anti-His antibody. As negative control, pcDNA3.1-FLAG empty vector replaced FLAG-ECRG1. Protein lysates of EC9706/FLAG-ECRG1+His-ECRG4 (Lane 1) and EC9706/FLAG+ His-ECRG4 (Lane 2) were immunoprecipitated by anti-FLAG antibody and visualized by anti-His antibody. The rabbit IgG antibody was used as negative control, and it showed no non-specific binding of ECRG1 with IgG. (C) EC9706 cells, transiently transfected with FLAG-ECRG1 and His-ECRG4, were immunoprecipitated by anti-His antibody and detected by anti-FLAG antibody. As negative control, pcDNA3.1-His empty vector replaced His-ECRG4. Protein lysates of EC9706/FLAG-ECRG1+His-ECRG4 (Lane 1) and EC9706/FLAG-ECRG1+His (Lane 2) were immunoprecipitated by anti-His antibody and visualized by anti-FLAG antibody. The rabbit IgG antibody was used as negative control, and it showed no non-specific binding of ECRG4 with IgG.