Trpv1 antibody

The kidney tissue was homogenized in 2 mL of 10 mmol/L Tris buffer (pH 7.6) containing 0.5 mmol/L MgCl₂, 50 mmol/L NaCl, and protease inhibitors. After spinning the kidney homogenates at 500 g for 5 min at 4°C, the supernatant was added with 10 μL of 0.5 mol/L EDTA and 100 μL of 10% Triton X-100, incubated on ice for 45 min and was spun at 22,000 g for 30 min at 4°C. The supernatant was electrophoresed at the protein concentration of 30 mg/mL on 7.5% SDS-PAGE gel and transferred to PVDF membranes (162-0180, Bio-Rad Laboratories, Hercules, California, USA). The TRPV1 antibody was from Neuromics (1:1000, RA10110, Neuromics, Edina, Minnesota, USA). The secondary antibody HRP-donkey anti-rabbit IgG was from Jackson (1:10,000, 711-035-152, Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania, USA). The immunoreactive bands were visualized by ECL reagents (RPN 2106, Amersham, GE Healthcare, Piscataway, New Jersey, USA) and the band intensity was quantified by using ImageJ (NIH, Bethesda, Maryland, USA). β-actin (1:2,000, sc-69879, Santa Cruz Biotechnology, Santa Cruz, California, USA) was used to normalize the protein loading.