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.