Fig. 2

Analysis of protein expression of autism-associated SERT coding variants. Impact of SERT coding variants on total and cell surface [¹²⁵I]RTI-55 binding. HeLa cells transiently transfected with hSERT or one of the SERT coding variants were subjected to intact cell binding assays at 4°C with the cocaine analog [¹²⁵I]RTI-55 (5 nM). A) Total binding values as defined with paroxetine (10 µM) as displacer. B) Surface labeling by [¹²⁵I]RTI-55 as defined with 5-HT (100 µM) as displacer. In vehicle-treated cells, hSERT total binding (fmol/10⁵) was 583.7 ± 20.6, and the surface binding was 175.7 ± 11.7. Results for A) and B) reflect mean values ± SEM of three separate experiments normalized to hSERT (100%). Binding levels were analyzed via a One-Way ANOVA followed by post-hoc Dunnett’s tests comparing mutant means to hSERT, with p < 0.05(*) taken as significant. C) Immunoblots of total cell extracts prepared from HeLa cells transfected with hSERT or one of the variants described in the study. D) Cell surface expression alterations in hSERT Gly56Ala, Ile425Leu, Phe465Leu and Leu550Val. Variants were transfected in parallel with hSERT into HeLa cells and cell surface transporters identified by immunoblotting of biotinylated samples. Quantitative estimations of relative E) total and F) surface density of hSERT, Gly56Ala, Ile425Leu, Phe465Leu and Leu550Val based on densitometry of biotinylation immunoblots. Data reflect mean values of three separate experiments ± SEM. Means were compared with a One-Way ANOVA followed by Dunnett’s test to compare variant surface expression to that achieved with hSERT, with p < 0.05 taken as significant (* Significantly elevated vs hSERT, p < 0.05; ⋕ Significantly reduced vs hSERT, p < 0.05). Figure reproduced from authors’ prior work [79]