Skin Protein Expression Analysis

Extracts of a skin sample or HaCaT cells were prepared and then centrifuged at 12,000 × g to precipitate the insoluble materials. The supernatant was retained and the concentration of protein in the supernatant was measured by a BCA Protein Assay Kit (Beyotime, Jiangsu, China). A sample of supernatant was resolved by SDS-PAGE using a 12% gel. The protein bands in the gel were transferred to a polyvinylidene difluoride (PVDF) membrane and blocked with 5% skim milk, followed by overnight incubation with anti-Filaggrin, anti-Involucrin, anti-PPARα, anti-PPARγ, anti-LXR (Proteintech, Chicago, IL, United States), or anti-ABCA1 (Absin, Shanghai, China). After that, the blot was washed and incubated with the appropriate secondary antibody (Cell Signaling Technology, Beverly, MA, United States). Finally, the blot was subjected to a detection assay using a chemiluminescence substrate (Pierce, Rockford, IL, United States), and images of the blot were acquired using an Amersham Imager (GE Healthcare Biosciences, Pittsburgh, PA, United States).