Trpm4

Cells were lysed using tissue protein extraction reagent (Thermo, USA) containing phosphatase inhibitor cocktail (1:100, Thermo), protease inhibitor cocktail (1:100, Thermo) and 5 mmol/L EDTA (Thermo), and lysates were oscillated and centrifuged (13 000 g, 15 minutes, 4°C). The supernatant was collected and stored at −80°C. The protein concentrations were determined using the BCA protein assay kit (Thermo). The protein was mixed with 5× SDS‐PAGE protein loading buffer (Beyotime, China) and denatured by 100°C water bath. Then, the samples denatured were electrophoresed in 10% SDS‐PAGE and transferred to PVDF membranes using transfer device (Bio‐Rad). The membranes were blocked with 5% non‐fat milk prepared in TBST for 1 hour at 37°C and then incubated at 4°C overnight with the primary antibodies: TRPC7 (1:1000, Santa cruz Biotechnology), TRPM4 (1:1000, Santa cruz Biotechnology), TRPC6 (1:1000, Santa cruz Biotechnology), TRPV2 (1:1000, Santa cruz Biotechnology) and the internal normalization mouse anti‐GAPDH (1:1000, Santa cruz Biotechnology). Next, the membranes were washed in TBST, incubated with secondary antibody: Goat anti‐Mouse IgG (H + L) IRDye 800CW or Goat anti‐Mouse IgG (H + L) IRDye 800CW (1:20 000; LI‐COR) for 1 hour at 37°C. The images were observed with a UVA Bio Imaging System and analysed with ImageJ software.

siRNA for CBS (hydroxylamine), CSE (dl-propargylglycine), TRPV1, TRPV3, TRPV6, and TRPM4 were purchased from Santa Cruz. H₂S donor NaHS was purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Capsaicin and capsazepine were purchased from EMD Millipore (Billerica, MA, USA). The concentrations of Capsaicin and capsazepine used for the treatment were 1 μM.