Western Blotting Analysis
Total protein was extracted from snap-frozen skin tissues or HaCaT cells using RIPA buffer (P0013J, Beyotime) containing 1% phosphatase and protease inhibitors. After centrifugation, clarified lysates were quantified using the BCA Protein Assay Kit (Thermo Scientific) and boiled with SDS loading buffer at 95 ℃ for 15 min. Then, we separated protein samples by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred them to polyvinylidene fluoride (PVDF) membranes (Amersham International), where 5% milk in TBST (TBS with 0.05% Tween 20) was used as the blocking buffer to block the membrane at room temperature for 1 h. Next, primary antibodies were diluted to a suitable concentration based on the manufacturer’s recommendations by dilution buffer (P0023A, Beyotime) and incubated overnight at 4 °C. After three washes in TBST, the protein samples were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 2 h. The protein bands were detected by the enhanced chemiluminescence (ECL) western-blotting substrate (Fdbio science, FD8000) and quantified using Image Lab (Bio-Rad).