Quantitative PCR
Total RNA was extracted from tissues and cells with TRIzol reagent (Tiangen Biotech, Beijing, CN), the absorbance of the extracted RNA was measured with a spectrophotometer (Bio–Rad Laboratories, West Berkeley, California, USA), and the integrity was examined by gel electrophoresis (Bio–Rad Laboratories, West Berkeley, California, USA). RNA with an A260/A280 ratio between 1.8-2.0 and three complete bands of 28S, 18S and 5S after 2% agarose gel electrophoresis was chosen for reverse transcription (Fig. 1A and B). Taq PCR Mastermix (Tiangen Biotech, Beijing, CN) was used for reverse transcription, and SYBR Green chemistry (Toyobo Co., LTD, Japan) was used for real-time PCR. Using the GAPDH gene as an internal reference, the reaction system and parameters were set according to the instructions. The internal reference and target gene primers were designed and synthesized by Shanghai Sangon Biological Co, Ltd (Table 2). Finally, the relative expression of each target gene was calculated by the following formula: 2-△△Ct (Ct value is the circulating domain value). The experiment was repeated three times.
Western blotting
The total proteins were extracted from the tissue and cell groups with RIPA buffer (Biyuntian Biotechnology Co., LTD, Shanghai, CN) containing the protease inhibitor phenylmethanesulfonyl fluoride (PMFF, Biyuntian Biotechnology Co., Ltd, Shanghai, CN). After the protein concentration was measured by a BCA protein assay kit (Biyuntian Biotechnology Co., LTD, Shanghai, CN) and the samples were denatured, 50 µg of protein per sample were separated by electrophoresis. Then, the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane, and the membrane was sealed in 5% skim milk powder dissolved in 0.5% Tween-TBS for 2 h at room temperature. After the membrane was washed, GAPDH (1:10000), TRPV1 (1:1000), SUR2B (1:500), Kir6.1 (1:1000) and eNOS (1:1000) primary antibodies were added and incubated overnight at 4 ℃ on a shaker. The next day, after the membrane was washed, horseradish peroxidase-labeled secondary antibodies (1:10000) were added and incubated at 37 ℃ for 2 h. Finally, the proteins were detected by chemiluminescence assays, GAPDH was used as an internal reference to analyze the optical density values of the target bands, and the ratio of the gray value of each target protein to GAPDH was used to calculate the relative expression level of the target protein. The experiment was repeated three times.