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Fig. 6
NECAB2 interacts with mGluR1 in vitro. (A) Overview of the protein interaction network derived from the Necab2 co-immunoprecipitation and subsequent mass spectrometry in the necab2+/+ and necab2–/– larvae. (B) REACTOME analysis revealed the significant biological processes detected in the necab2+/+ larvae compared to the necab2–/– larvae. (C) Gene Ontology (GO) analysis revealed the significant biological processes detected in the necab2+/+ larvae compared to the necab2–/– larvae. (D–D″′) NECAB2 interacts with mGluR1 through the NHR domain uncovered by co-immunoprecipitation analysis. The HEK293 cells were transiently transfected with mGluR1-mCherry plus NECAB2-Myc (D), EGFP-EF (D′), EGFP-NHR (D″), or EGFP-ABM (D″′) for lane 1 and lane 3 respectively, and indicated vectors plus mCherry-vector for lane 2 and processed for immunoprecipitation using mouse anti-Myc antibody (D) or anti-EGFP antibody (D–D″′), with normal mouse IgG for negative control in lane3. The crude extracts (Input) and immunoprecipitations (IP) were analyzed by SDS-PAGE and immunoblotted using the rabbit anti-mCherry antibody (D–D″′), mouse anti-Myc antibody (D) or mouse anti-EGFP antibody (D′–D″′). (E–E″′) NECAB2 has self-interaction through either NHR or ABM domain uncovered by co-immunoprecipitation analysis. The HEK293 cells were transiently transfected with NECAB2-Flag plus EGFP-NECAB2 (E), NECAB2-Flag plus EGFP-EF (E′), NECAB2-Flag plus EGFP-NHR (E″), or NECAB2-Flag plus EGFP-ABM (E″′) for lane 1 and lane 3, and the indicated vectors plus EGFP-vector for lane 2 and processed for immunoprecipitation using mouse anti-Flag antibody, with normal mouse IgG for negative control in lane 3. The crude extracts (Input) and immunoprecipitations (IP) were analyzed by SDS-PAGE and immunoblotted using mouse anti-EGFP antibody (E–E″′) or mouse anti-Flag antibody (E–E″′). (F–F″) Co-immunoprecipitation analysis of NHR and ABM domains showed self-interaction but no cross-interaction of the two domains. The HEK293 cells were transiently transfected with Flag-NHR plus EGFP-NHR (F), Flag-ABM plus EGFP-ABM (F′), and Flag-ABM plus EGFP-NHR (F″) for lane 1 and lane 3, and indicated vectors plus EGFP-vector for lane 2 and processed for immunoprecipitation using the mouse anti-Flag antibody, with normal mouse IgG for negative control in lane3. The crude extracts (Input) and immunoprecipitations (IP) were analyzed by SDS-PAGE and immunoblotted mouse anti-Flag antibody (F–F″) or mouse anti-EGFP antibody (F–F″).