Fig 1: Mutants R57C, R57G, R132C, and Y133S lose interactions with partner phosphoprotein in vitro and in cells. (A) A novel in vitro BRET assay developed in this study to assess the interaction between recombinant 14-3-3γ (C-terminally tagged with nanoluc (nLuc; BRET donor)) and partner phosphopeptide (N-terminally tagged with FITC; BRET acceptor). Upon the addition of furimazine, substrate for nLuc, light emitted by nLuc excites FITC in the close proximity (<10 nm), which occurs when FITC-phosphopeptide bound to 14-3-3γ-nLuc. (B) The production and purification of recombinant 14-3-3γ-nLuc from E. coli showed similar expression levels and purity between wild-type and mutants 14-3-3γ-nLuc. (C–E) Recombinant 14-3-3γ-nLuc (1 nM) was mixed with increasing doses of FITC-pTH (C), FITC-ppLRRK2 (D), or FITC-pSLP76 (E) before the addition of 250 nM furimazine. Wild-type 14-3-3γ-nLuc exhibits increased BRET signal in a dose-dependent manner, while mutants 14-3-3γ-nLuc show significantly decreased BRET signals indicating their incapability to efficiently bind FITC-phosphopeptides. (F–H) Cellular interactions of 14-3-3γ with TH or SLP76 as determined by IP. N2a cells were co-transfected with wild-type or R57C mutant 14-3-3γ-GFP and 3xHA-TH. IP of 14-3-3γ was performed using a nanobody against GFP, and the co-precipitation of TH was analyzed by SDS-PAGE and Western blot. Antibody against GFP was used for the detection of 14-3-3γ and against HA for TH (F). N2a cells were co-transfected with GFP-SLP76 and wild-type or R57C mutant 14-3-3γ-3xHA. IP of SLP76 was performed using a nanobody against GFP, and the co-precipitation of 14-3-3γ was analyzed by SDS-PAGE and Western blot. Antibody against GFP was used for the detection of SLP76 and against HA for 14-3-3γ (G). Quantification of the coimmunoprecipitation between 14-3-3γ and TH or SLP76 (H). Data in the panels (C–E,H) represent mean ± SEM (n ≥ 3). Statistical analysis for panel H was performed using one sample t-test, **** p < 0.0001.