Removal of 14-3-3 activates RGS18 function. HEK293T cells were transfected with the mCherry-tagged RGS18 constructs S216A or S49A/S218A or the pmCherry vector as a control. Cells were then stained with 0.5μM Fluo-4, a Ca²⁺ dye, for 25 minutes at 37°C. Ca²⁺ responses and mCherry transfection efficiency measurements by flow cytometry were performed using an Accuri C6 flow cytometer. Fluo-4–stained single cells that contained Cherry constructs were visualized using a 488-nm laser and 530 ± 15/675LP filter combination. Cells were stimulated with 0.1 U/mL of thrombin (arrow) and intracellular Ca²⁺ changes were monitored as changes in Fluo-4 fluorescence. Data from 4 independent experiments performed in triplicate were normalized as indicated in “Methods” and are shown as means ± SEM. Thrombin stimulation was carried out at time point 1.0 minutes. Measurements taken at time points 1.2 and 1.4 minutes are shown as separate bar charts (bottom panels). Statistical analyses of data were performed using 1-way ANOVA in combination with the Bonferroni posttest. ****P < .0001; ***P < .001; **P < .01; *P < .05. mCherry-S216A-RGS18– and mCherry-S218/49A-RGS18–transfected cells display a significantly lower increase in Ca²⁺ compared with mCherry-transfected control cells (time point 1.2 minutes, P < .001 and P < .0001, respectively; time point 1.4 minutes, P < .01 and P < .0001, respectively). Comparing 14-3-3–deficient RGS18 (mCherry-S218/49A-RGS18) with 14-3-3–bound RGS18 (mCherry-S216A-RGS18), Ca²⁺ peaks are significantly lower in cells transfected with 14-3-3–deficient RGS18 (time point 1.2, P < .01; time point 1.4, P < .05).