Fig. 3.

Signaling properties of 5-HT₆ receptor mutants. IMCD3 cells were transfected with empty vector ± 5-HT₆R plasmid (WT-5-HT₆R, 5-HT₆R^D106A, 5-HT₆R^del406–411) and treated with vehicle, 1 μM WAY-208466 (agonist) or 1 μM SB-399885 (antagonist), or 1 μM concentration of both drugs before cell lysis and tissue harvest for cAMP assay. (A) Absolute cAMP levels and (B) cAMP relative to corresponding vehicle controls are shown after 10 minutes of drug treatment. (C and D) HEK 293 were transfected with empty vector ± 5-HT₆R plasmid or mutant receptors and treated with vehicle or 1 μM WAY-208466 for 15 minutes. Immunoprecipitation from cell lysates was conducted using anti-Fyn Ab, bound material was eluted, subjected to SDS-PAGE, and then immunoblotted for Fyn and phosphorylated Fyn (p-Y420). (C) Representative Western blot of IP-isolated Fyn and Fyn phosphorylated at Y-420. (D) Graph depicting p-Y-420 intensity/Fyn intensity. One-way ANOVA, n = 3 biologic replicates for each experiment. Bonferroni post-hoc. *P < 0.05. ^$P < 0.01 compared with receptor vehicle control.