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. 2017 Jan 10;112(1):87–98. doi: 10.1016/j.bpj.2016.11.3197

Figure 6.

Figure 6

Multiplexing measurements of TRPV activity using multicolor BRET. (A) Example of a three-color BRET spectrum and its decomposition, measured in a coculture containing three HEK293T subpopulations transfected with aquamarine-Luc, mAmetrine-Luc, or LSSmOrange-Luc. (Gray dots) Experimental data. (Red line) Best fit to the experimental data. (Purple, blue, green, and orange lines) Spectral components of Luc, aquamarine, mAmetrine, and LSSmOrange, respectively. (B–D) Multicolored BRET produced by Luc and aquamarine, Luc and mAmetrine, and Luc and LSSmOrange were measured in real time in one sample containing a mixed population of cells expressing Luc-TRPV3/aquamarine-CaM (blue line, basal BRET of 0.027 ± 0.002), TRPV1-Luc/mAmetrine-CaM (green line, basal BRET of 0.015 ± 0.001), and TRPV4-Luc/LSSmOrange-CaM (red line, basal BRET of 0.017 ± 0.001) constructs. One mM Drofenine (B), 20 μM CAPS (C), or 100 nM GSK101 (D) were injected 75 s after the beginning of the experiment initiated by the injection of purple coelenterazine into the buffer. Results represent the mean of three independent experiments.