Extended Data Fig. 3: Photostability and intracellular signaling couplings of GRABeCB2.0 sensor.
From: A fluorescent sensor for spatiotemporally resolved imaging of endocannabinoid dynamics in vivo
a, Normalized fluorescence of EGFP-CAAX and eCB2.0 (in the absence and presence of 2-AG) in HEK293T cells during 1 P (confocal) bleaching. b, Integrated fluorescence of EGFP-CAAX and eCB2.0 (in the absence and presence of 2-AG) shown in a; n = 29, 27, 28 cells from 3 cultures. Boxes show the first and third quartiles as well as the median (line), and the whiskers extend to the most extreme data point that is no more than 1.5× the interquartile range from the box. Two-tailed Mann-Whitney tests were performed: P = 1.44E-10 (between EGFP and eCB2.0 in saline) and 1.37E-6 (between EGFP and eCB2.0 with 2-AG). c, Fast and slow time constants and slow component amplitudes of EGFP-CAAX and eCB2.0 (in the absence and presence of 2-AG) traces fit by double exponentials. d, Normalized fluorescence of EGFP-CAAX and eCB2.0 (in the absence and presence of 2-AG) in HEK293T cells during 2 P bleaching. e, Time constants of EGFP-CAAX and eCB2.0 (in the absence and presence of 2-AG) traces fit by single exponentials; n = 79, 48, 104 cells from 3 cultures. Boxes show the first and third quartiles as well as the median (line), and the whiskers extend to the most extreme data point that is no more than 1.5× the interquartile range from the box. Two-tailed Mann-Whitney tests were performed: P = 0.0049 (between EGFP and eCB2.0 in saline) and 0.0581 (between EGFP and eCB2.0 with 2-AG). f, Schematic diagram depicting the strategy for measuring G protein activation using the chimeric Gαq-i protein. g, Representative traces showing the jRGECO1a responses to 2-AG perfusion in cells expressing CB1R, CB1R + eCB2.0 or eCB2.0. h, Dose-response curves of peak jRGECO1a ΔF/F0 measured in cells expressing CB1R, CB1R + eCB2.0, or eCB2.0; n = 4, 4 and 3 cultures, mean ± s.e.m. Two concentrations of 2-AG were used for eCB2.0 expressed cells. Two-tailed Student’s t tests were performed: P = 0.2392, 0.1455, 0.6711, 0.9191 and 0.8371 (between CB1R and CB1R + eCB2.0); P = 0.0156 and 0.0015 (between CB1R and eCB2.0). i, G protein coupling was measured using a BRET Gβγ sensor in cells expressing CB1R, eCB2.0, or eCBmut; n = 3 experiments, mean ± s.e.m. Two-tailed Student’s t tests were performed: P = 3.84E-05, 0.4082, 0.0699 and 0.2961. j, β-arrestin coupling was measured using the Tango assay in cells expressing CB1R, eCB2.0, or eCBmut; n = 3 wells each, mean ± s.e.m. k, Dose-response curves of eCB2.0 to 2-AG measured in cells expressing eCB2.0 or eCB2.0 + CB1R; n = 3 wells each, mean ± s.e.m. Two-tailed Student’s t tests were performed: P = 0.3036, 0.3231, 0.7697, 0.7900, 0.9723, 0.5482 and 0.1383. ***, p < 0.001; **, p < 0.01; *, p < 0.05; n.s., not significant.
