2.5 Western blot
Total proteins were isolated from tissues using a total protein
extraction kit (Keygen, Nanjing, China). A total of 40 µg protein was
separated using SDS-PAGE and transferred onto polyvinylidene difluoride
(PVDF) membranes and then blocked with 5% fat-free milk at room
temperature for 2 h. The immune-blot was incubated with primary antibody
(1:1000 dilution; Cell signaling) and GAPDH (1:1000 dilution; Cell
signaling) was used as a control. The signals were detected using a
Super ECL Plus Kit (Keygen) and determined by quantitative analysis
using UVP software (UVP, LLC, Upland, CA, United States).