2.11. Western Blot Assay

Protein levels were monitored in the skin by homogenizing isolated skin tissue in cold lysis buffer at 3650 rpm, 6.00 m/s linear speed, and 6 cycles for 30 s (Bioprep-24R, Allsheng, Hangzhou, China). The samples were centrifuged at 13,500 rpm for 10 min at 4 °C. Protein concentrations in the lysate were determined by BCA assay (Thermo Fisher Scientific Inc.) and the extracted protein was separated by SDS-PAGE. Antibodies against aldehyde dehydrogenase 1 (ALDH1) (Santa Cruz Biotechnology, Dallas, TX, USA), α-tubulin, β-catenin, pyruvate kinase M2 (PKM2, Cell Signaling Technology, Danvers, MA, USA), and cyclin D1 (Merck, Kenilworth, NJ, USA) were detected using horseradish peroxidase (HRP)-conjugated secondary antibody (Thermo Fisher Scientific Inc.) and the Immobilon Western Chemiluminescent HRP Substrate Kit (Millipore, Billerica, MA, USA). The density of the band was measured to obtain the relative density using loading controls. Values are expressed as arbitrary densitometric units that correspond to signal intensity.