Fig. 4: WT and R919* TRPA1 co-localize at the plasma membrane in cells.

a, b Surface biotinylation analysis of 3×FLAG-WT or R919* hTRPA1 or FLAG-Torsin A from transfected HEK293T cells. Biotinylated proteins were precipitated by Neutravidin resin pulldown and probed using HRP-conjugated anti-FLAG antibody (a) and quantified (b). Tubulin from whole cell lysates (10%, inputs) was the loading control. Torsin A and Tubulin were negative controls for relative plasma membrane localization. Full blots are included in Supplementary Fig. 14. b Expression of WT (black) or R919* (pink) hTRPA1 in the plasma membrane is represented by filled bars (corresponding to lanes 2 and 3 in a, respectively). Expression of WT or R919* hTRPA1 in the plasma membrane from co-transfected cells is represented by striped bars (corresponding to lane 4 in a). Data represent mean ± SEM. **p < 0.01, n.s. not significant. n = 6 independent experiments, two-tailed Student’s t-test. c Representative deconvolved immunofluorescence images of HEK293T cells expressing GFP-R919*, 3×FLAG-WT, or GFP-R919* and 3×FLAG-WT hTRPA1. Cells were stained with anti-GFP (green) and anti-FLAG (red) antibodies. Plasma membrane was labeled with wheat germ agglutinin (blue). Scale bar indicates 2 µm. Images are representative of 3 independent experiments. d Quantification of GFP-R919* hTRPA1 fluorescence intensity in the plasma membrane of HEK293T cells relative to the cell interior (arbitrary units, arb. units). Values were obtained using line-scans of raw images. Data represent mean ± SEM. *p < 0.05, n = 30 cells per condition, two-tailed Student’s t-test. e Representative deconvolved immunofluorescence images of HEK293T cells co-expressing 3×FLAG-WT hTRPA1 with GFP-WT or GFP-R919* hTRPA1. Cells were stained with anti-GFP (green) and anti-FLAG (red) antibodies. Scale bar indicates 2 µm. Images are representative of 3 independent experiments. f Scatter plot of 3×FLAG-WT hTRPA1 and GFP-WT (left) or R919* hTRPA1 (right) fluorescence intensity at the plasma membrane of HEK293T cells. Pearson’s correlation coefficients (r) were determined using raw images and the coefficient of determination (r²) is depicted in the lower right corner of each plot. A line of best fit is shown in red. p < 0.0001, n > 450 pixels in 1 cell per condition. b, d, f Source data are provided as a Source Data file.