B-IA covalently binds reactive groups in hTRPA1. (A) Representative images of B-IA binding (green) on immunoprecipitated hTRPA1 quantified by the antibody against V5 tag (red). V5-tagged hTRPA1 had an expected mass of 131 kD. Overlay shows B-IA binding colocalizes with TRP channel. (B) Mean ± SEM for normalized B-IA binding on hTRPA1 (n ≥ 3). (C) Normalized 10 µM B-IA binding to hTRPA1 (60 s) is reduced by pretreatment with unlabeled IA (100 µM, 10 min) but not by TRPA1 inhibitors HC030031 (30 µM) and AP-18 (30 µM; n ≥ 3). The asterisk denotes difference from control (*, P < 0.05). (D) Mean ± SEM loss of 412-nm absorption after the reaction of 5 µM TNB with 100 µM IA (10 min) and 1 mM NEM (n = 3). (E) Normalized 10 µM B-IA binding to hTRPA1 (60 s) is unaffected by removal of intracellular polyphosphates (saponin) or by polyphosphate supplementation (saponin + NaPPPi; n = 3). (F) Normalized 10 µM B-IA (60 s) binding to hTRPA1, mTRPA1, rsTRPA1, and hTRPV1 (n ≥ 3). Asterisks denote difference from control (clear bars) with IA pretreatment (100 µM, 10 min; hatched bars; *, P < 0.05). (C, E, and F) Error bars denote SEM. All bands in the blots are 131 kD, except for the yellow bars in F, which are 90 kD.