Fig. 5: TRPA1 and CaM co-localize in cells.

a Representative deconvoluted Airyscan images of Neuro2A cells co-expressing 3xFLAG-WT (left) or Δ1109–1119 hTRPA1 (right) with V5-WT CaM. Cells were stained with anti-FLAG (red) and anti-V5 (green) antibodies. Scale bar indicates 2 µm. Images are representative of 3 independent experiments. b Pearson’s correlation coefficients (R) were determined for WT hTRPA1:WT CaM or Δ1109–1119 hTRPA1:WT CaM using raw images (see Supplementary Fig. 6). Data represent mean ± SD. n = 41 (WT) or 40 (Δ1109–1119) cells from 3 independent experiments, two-tailed Student’s t-test. n > 10,000 pixels in 1 cell per condition. c Proximity biotinylation approach. TurboID (orange star) fused to the CaM C-lobe (green circle) is co-expressed with hTRPA1 variants (grey) in cells. Addition of biotin (pink star) to the media will facilitate generation of a local reactive biotin cloud (purple) that will biotinylate TurboID-CaM C-lobe and TRPA1, pending an interaction. d Immunoblotting analysis of biotinylated 3xFLAG-WT, Δ1109–1119, or W1103A hTRPA1 co-expressed with empty vector (-), 3xFLAG-TurboID-CaM C-lobe, or 3xFLAG-TurboID in HEK293T cells. FLAG immunoprecipitated eluates were probed for biotinylation with Streptavidin-HRP. FLAG-tagged proteins were probed using an anti-FLAG antibody. Tubulin was the loading control. Blots are representative of four independent experiments. e Quantification of the percent change to TRPA1 biotinylation with TurboID-CaM C-lobe versus TurboID from experiments as in (d). Pulldown Streptavidin-HRP (e.g., biotinylation) was normalized to FLAG and Tubulin from inputs for each sample. Biotinylation by TurboID was subtracted from biotinylation by TurboID-CaM C-lobe. Data are presented as the percent change from biotinylation with TurboID. Data represent mean ± SEM. n = 4 independent experiments, one-way ANOVA with Bonferroni’s post hoc analysis. b, e P-values indicated in figure panels.