(A–D) Average traces from calcium imaging experiments with HEK cells transiently transfected with zebrafish trpa1b (n = 85) (A), zebrafish trpa1b + zebrafish tlr7 (n = 80) (B), mouse Trpa1 (n = 134) (C), or mouse Trpa1+ mouse Tlr7 (n = 89) (D). n = 22, 23, 10, and 19 untransfected control HEK cells per experiment, respectively. No gross differences were observed between the two conditions for each species. (E) Quantification of peak fluorescence intensity achieved during stimulation with 100 μM IMQ across all HEK cell transfection conditions. In all species examined, no significant difference was observed between cells transfected with only Trpa1 and cells transfected with Trpa1 plus the corresponding Tlr7. (F–H), Immunohistochemistry performed on HEK cells transfected with pIRES-eGFP only (F), mouse Tlr7 (G), or human TLR7 (H). As shown, TLR7 labeling (red) was only observed HEK cells transfected with Tlr7 constructs. (I–J), I/V curves from voltage clamp experiments using cells transfected with mTRPA1 (I) and mouse Trpa1 + mouse Tlr7 (J). As shown in (I), 100 μM IMQ elicited greater current influx in transfected cells than under basal conditions, an effect that disappeared during washout. Cells transfected with both mouse Trpa1 and mouse Tlr7 also showed significant current flux during IMQ stimulus, but this was not different from cells transfected only with mTRPA1. n = 5 cells per condition. (K) a table of EC50 values for both IMQ and AITC stimuli in cells transfected with zebrafish trpa1b, mouse Trpa1, and human TRPA1 in Figure 3D–F. As shown, the EC50 values for IMQ are much greater than those of AITC for all species of TRPA1. ΔF/F (fluorescence intensity change) is expressed as a normalized 340 nm/380 nm intensity ratio. (*p<0.05, **p<0.01, ***p<0.001, Student’s t-test. Bars are expressed as means ± s.e.m.