##Western blot
Kidneys were collected and placed in a solution (300 ℃) containing with
20 mM of Tris pH 7.4, 50 mM of NaCl, 1% Triton X-100, and protease
inhibitor 10 μL for homogeneous cracking in cracking liquid. Protein
samples were taken out at –80 ℃ and refrigerated to reduce protein
degradation. The sample was placed in sodium dodecyl sulfate gel loaded
buffer solution, boiled for 5 min and centrifuged at 4 ℃ at 6,000 rpm
for 3 min, after which the supernatant was collected. The denatured
protein underwent electrophoresis in gel and was then transferred to the
polyvinylidene fluoride membrane for 2 hours at 65 V and 4 ℃ through a
transfer device. After blocking on a shaking table at room temperature
was conducted for 2 hours, the first antibody was incubated at 4 ℃
overnight. This was followed by 3 washes with Tris-buffered saline with
Tween. After 10 minutes washing, goat anti-rabbit immunoglobin G
combined with horseradish peroxidase (1:2000; Sigma-Aldrich) was added
and incubated for 1.5 h. The membrane was washed with TBST and observed
with an enhanced chemiluminescence system.