(A) The addition of CFP and YPF tag to the N-&-C terminus respectively, does not impact the ability of SERT Ala56 to enhance [3H] 5-HT uptake (20 nM for 10 min in transfected CHO cells transfected at 37°C), one-way ANOVA followed by Tukey’s post-hoc test, *P ≤ 0.05, n= 6-9; Error bars represent ± SEM. (B) Representative FRET images of transfected CHO cells with indicated CFP-YFP dual tagged constructs with CFP excitation/emission, YFP excitation/emission, calculated FRET efficiency (CFP excitation/YFP emission). (C) FRET efficiency, calculated as described in Methods. (Error Bars ± SEM, One-way ANOVA followed by Tukey’s post hoc test, *P ≤ 0.05, n=22). (D) Histogram of FRET efficiency show C-Ala56-Y distribution is significantly shifted to the right compared to WT (Kruskal-Wallis ANOVA followed by Dunn’s post hoc analysis, P ≤ 0.05, n=22) and C-Asn605-Y split into two Gaussian’s curves.