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).