Fluorescence emission spectra from cells expressing L-ANAP–incorporating TRPV1 channels. (A–C) Emission spectra for unroofed cells transfected with pANAP and TRPV1-Q169TAG-YFP (A), TRPV1-K240TAG-YFP (B), or TRPV1-S404TAG-YFPv and cultured with L-ANAP-ME. Spectra were acquired as described in Fig. S2 but using the excitation cube appropriate for ANAP with the band-pass emission filter replaced by a 405-nm long-pass emission filter. The red traces represent the mean emission spectra from three unroofed cells, normalized to their peak before averaging. The black traces are from L-ANAP-ME conjugated to beads, also shown in Fig. S2 B. The dashed trace in A represents a fit to the cell data (red) by the sum of the L-ANAP-ME spectrum from the beads and a spectrum of YFP (blue), measured separately.