N methylmaleimide

Cells were homogenized by scraping into 90 µl Urea/SDS buffer (10 mM Tris‐HCl pH 6.8, 6.7 M urea, 1% w/v SDS, 10% v/v glycerol and 7.4 µM bromophenol blue, containing 50 µM phenylmethysulfonyl fluoride and 50 µM N‐methylmaleimide, all Sigma Aldrich). Lysates were sonicated and 10% v/v β‐mercaptoethanol (Sigma Aldrich) added. Proteins were separated by SDS‐PAGE using 10% polyacrylamide gels; 1.5 M Tris, 0.4% w/v SDS, 10% acrylamide/bis‐acrylamide (all Sigma Aldrich), electrophoresis buffer; 25 mM tris‐HCl, 190 mM glycine, 0.1% w/v SDS, pH 8.5 (all Sigma Aldrich). Proteins were transferred to a nitrocellulose membrane using the TurboBlot^™ system (BioRad) and blocked at room temperature in Odyssey® TBS‐Blocking Buffer (Li‐Cor BioSciences) for 1 h. The membranes were probed overnight at 4°C diluted in blocking buffer, washed 3 × 5 min in PBS with 0.1% Tween (both Sigma Aldrich) and probed for 1 h at room temperature in the dark with IRDye® conjugated secondary Abs against goat IgG (800 CW) and rabbit IgG (680 CW), raised in goat or donkey (LiCor BioSciences), diluted in Odyssey® TBS‐Blocking Buffer at 0.05 µg/ml. Membranes were then washed for 3 × 5 min in PBS with 0.1% Tween, rinsed with ddH₂O and imaged using the Odyssey® CLX 9120 infrared imaging system (LiCor BioSciences). Image Studio Light v.5.2 was used to process scanned membranes.

For inhibition of TRPA1, we incubated cells with the antagonist HC-030031^{13 (link)} (40μM in DMSO; Cayman Chemicals #11923) for 45 min before stimulation. For activation of TRPA1, we used NMM^{13 (link)} (N-Methylmaleimide; 100μM in DMSO Sigma-Aldrich #389412) or AITC^{28 (link)} (30μM in DMSO, allyl isothiocyanate; Sigma-Aldrich # 377430). For activation of Piezo1, we used yoda-1⁷⁵ (10 μM in DMSO; Tocris #5586). For activation of TRPV1 we used capsaicin^{76 (link)} (3 μM in DMSO; Sigma-Aldrich #M2028). The final concentration of DMSO in the external solution was 0.1% or lower for all groups, which was also used as vehicle control. For cytoskeleton experiments, nocodazole (5μM; Tocris, #1228), jasplakinolide (200 μM; ThermoFisher # J7473), paclitaxel (600 nM; Sigma-Aldrich # T7191), cytochalasin D (5μM; Cayman Chemicals, #11330) or latrunculin A (1 μM; Cayman Chemicals, # 10630) in 0.1% DMSO were added to the culture media 45 min prior to imaging^{37 (link)}. For pharmacology in primary neurons, we used TRPV1 antagonist, A784168^{77 (link)} (20μM; Tocris, #4319, 45 min incubation) in 0.1% DMSO, BAPTA^{78 (link)} (30μM; Invitrogen, #B1204, 45 min incubation) directly dissolved in culture media and TTX^{79 (link)} (18μM; tetrodotoxin citrate; Tocris #1069, 5 min incubation, where we also inhibit TTX-R channels).