Extended Data Fig. 6: Activation of TAS2R14 by cholesterol and cmpd28.1.
From: Bitter taste receptor activation by cholesterol and an intracellular tastant
a,b, Basal activities of TAS2R14 coupled with Ggust (a) and Gi1 (b) measured by BRET2 assays. Data represent mean ± s.e.m. of n = 3 biological replicates. The activation levels according to the increase of relative amounts of transfection of TAS2R14 were normalized by BRET ratio from the pcDNA3.1-transfected conditions. c-g, Split luciferase biosensor cAMP inhibition assay upon cholesterol depletion and replenishment. The values represent the mean ± s.e.m. of n = 3–6 biologically independent experiments. Significant differences were determined by ordinary one-way ANOVA with Dunnett’s test for multiple comparison analysis with reference to the pcDNA3.1 (c,d) and WT (e-g). h-j, BRET2 assays for TAS2R14 mutations at the orthosteric binding pocket in the absence/presence of cmpd28.1. Data represent mean ± s.e.m. of n = 3 biological replicates. h,i, All values are subtracted by net BRET of pcDNA3.1 in each of the six cellular conditions, separately. j, The net BRET values were subtracted by the net BRET of WT in the absence of cmpd28.1. Statistical differences were determined by ordinary two-way ANOVA with Dunnett’s test for multiple comparison analysis with reference to each of WT with either 0 μM or 10 μM cmpd28.1, separately. k-n, Agonist-stimulated TAS2R14 (k) and TAS2R46 (m) activation upon conditions of cholesterol depletion and replenishment. Representative column graphs for TAS2R14 (l) and TAS2R46 (n) adopted from the values at 100 μM agonists in a dose-response curve for TAS2R14 (k) and TAS2R46 (m), respectively. Data represent mean ± s.e.m. of n = 6 (k, l) and 3 (m, n). Statistical differences were determined by ordinary one-way ANOVA with Dunnett’s test for multiple comparison analysis with reference to the no-treatment condition (l, n). o-q, BRET2 assays with a dose-response of cmpd28.1 under the conditions of no treatment (o), depletion (p), and replenishment (q). All the significant differences were indicated by ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05, and ns (not significantly different) (c-g, j,l,n).