5.9. Western blot analysis

Protein extracts from both dorsal skin tissue and HaCaT cells were obtained using PRO-PREP™ protein extraction solution (Intron Biotechnology, Seoul, Korea), followed by homogenization at 4 °C. Afterward, the samples were then centrifuged at 11,000×g for 30 min at 4 °C to remove cellular debris, and the supernatant was quickly frozen. As per the manufacturer's protocol, Bio-Rad protein assay reagent was utilized to measure the protein concentrations. Proteins from each sample were separated by electrophoresis on an 8%–12 % SDS-polyacrylamide gel and then transferred onto a polyvinylidene difluoride (PVDF) membrane via electroblotting. For 30 min, the membranes were blocked with 2.5 % skim milk solution at room temperature, followed by overnight incubation with a primary antibody (1:1000 dilution in Tween 20/Tris-buffered saline (T/TBS)) at 4 °C. Following three washes with T/TBS, the blots were treated with a horseradish peroxidase-conjugated secondary antibody (1:2000 dilution) for 2 h at room temperature. The blots were washed again three times with T/TBS and visualized using an enhanced chemiluminescence system (GE Healthcare, WI, USA). Densitometric analysis was performed using Bio-Rad Quantity One software (version 4.3.0; Bio-Rad Laboratories, Inc.).