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).