Fig. 2. TrkB interacts with mGlu₂ but not with mGlu₃.

(A) Western blots of anti-Flag immunoprecipitates obtained from HEK-293 cells expressing human Flag-tagged mGlu₂ or mGlu₃ in the absence or presence of human TrkB. Inputs (lysate) and anti-Flag immunoprecipitates (IP) were probed with anti-Flag antibody to detect mGlu₂ and mGlu₃, anti-TrkB antibody, and with anti-GAPDH antibody. The data are representative of four independent experiments performed on different cultures. (B) Representative images of PLA experiments performed with anti-Flag and anti-TrkB antibodies in HEK-293 cells expressing either human Flag-tagged mGlu₂ or mGlu₃ in the absence or presence of human TrkB. PLA signal is shown in yellow, cell nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI) in blue, and green fluorescent protein transfection control is shown in magenta. Scale bars, 5 μm. Data are representative of four independent experiments performed on different cultures. (C) Representative images of PLA experiments performed with the anti-mGlu₂ nanobody and the anti-TrkB antibody on OB, PFC, DG, and CB slices from wild-type (WT) or mGlu₂ KO (mGlu₂^−/−) mice. PLA signal is shown in yellow; cell nuclei are stained with DAPI in blue. Scale bars, 50 μm. The histogram shows means ± SEM of total PLA punctae per region of interest from 10 to 13 mice per condition. Two-way analysis of variance (ANOVA) (compared between wild type and mGlu₂^−/−, post hoc Šidák’s test): ***P < 0.001. (D) BRET saturation curves obtained from HEK-293 cells expressing a fixed amount of Rluc-TrkB or Rluc-mGlu₅ and increasing amounts of Venus-mGlu₂ or Venus-mGlu₃ or Venus-mGlu₂C3 (mGlu₂ with mGlu₃ C-terminal) or membrane bound CAAX-Venus or cytoplasmic Venus. Curves show means ± SEM of net BRET signals measured in three technical replicates of one experiment representative of four biological replicates. Two-way ANOVA (post hoc Šidák’s test): F_8,26 = 638.6, ***P < 0.001.