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

Figure 2.

Figure 2

Measuring TRPV1 activity using an intermolecular BRET probe. (A) Schematic representation of the intermolecular BRET between TRPV1 and Calmodulin. YFP was fused to the N-terminal of Calmodulin and Luc to the C-terminal of TRPV1. After activation of TRPV1, the distance d between Luc and YFP was expected to be modified. (B) Kinetic measurement of the effect of 20 μM CAPS on cells expressing TRPV1-Luc and either YFP-CaM WT or YFP-CaM1234, pretreated or not with 10 μM BAPTA-AM for 30 min before activation with CAPS. (Star) Time of CAPS injection. Data represent one out of three independent experiments. (C) Titration assays using HEK293T cells transfected with increasing amounts of YFP-CaM WT (circles), YFP-CaM1234 (diamonds), or unfused YFP (square) and a fixed amount of TRPV1-Luc. Transfected cells were activated (open symbols) or not (solid symbols) with 20 μM CAPS before BRET reading. Results represent the data obtained over three independent experiments performed in duplicate. To see this figure in color, go online.