4.4. Western Blot Assay

Western blotting was used to analyze the changes in the various proteins in cells. HaCaT cells were seeded in a 3.5 cm culture dish. After the cells reached 90% confluence and were starved for 24 h, they were pretreated with neferine for 20 min and then stimulated with TNF-α/IFN-γ for 30 min or 1 h, respectively. After scraping, the cells were pulverized by ultrasound and centrifuged (13,200 rpm, 10 min, 4 °C). After centrifugation, the supernatant was taken and the protein was quantified with a Pierce protein assay kit (Pierce, Rockford, IL, USA). About 20–40 μg of protein was electrophoresed on 10% SDS-polyacrylamide gel and then electroporated with a PVDF membrane. After the transfer was completed, the PVDF membrane was put into TBS-T (Tris-buffered saline/0.05% tween 20) solution containing 5% skimmed milk powder and shaken for 1 h to avoid non-specific binding. Then, the PVDF membrane was washed with TBS-T three times (10 min each time). Then, primary antibodies (in a 1:1000 dilution) were added. The PVDF membrane was left overnight at 4 °C and then washed three times with TBS-T for 10 min each time. After adding secondary antibodies (diluted to 1:1000) for 1 h, the PVDF membrane was washed three times with TBS-T for 10 min each time. Finally, the developer was added, and the membrane was placed in a chemical luminescence extraction system (BIOSTEP Celvin^®) for shooting.