Proteinase and phosphatase inhibitor cocktail

Protein was isolated from cells using cold lysis buffer containing a proteinase and phosphatase inhibitor cocktail (Beyotime, Shanghai, China). The protein concentrations in the supernatants were measured using a BCA protein quantification kit (Beyotime, Shanghai, China). Samples containing equal amounts of protein (20 μg) were separated by 10% SDS-polyacrylamide gel electrophoresis and then transferred to a polyvinyldifluoride membrane (Bio-Rad Co. USA). The membranes were blocked and then incubated overnight at 4 °C with primary antibody (PERK, Santa Cruz Biotechnology, catalogue no.sc-377,400; phospho-PERK (p-PERK), Abcam, catalogue no. ab192591; eIF2α, Cell Signaling, catalogue no.5324; phospho-eIF2α (p-eIF2α), Abcam, catalogue no. Ab32157; caspase9, Cell Signaling, catalogue no.9504; caspase3, Cell Signaling, catalogue no. D3R6Y; β-actin, Cell Signaling, catalogue no. D6A8), washed four times using TBST (5 min each time). Then, the membrane was incubated with the corresponding HRP-conjugated secondary antibody at 25 °C for 1 h, washed four times with TBST for 20 min, and visualized using ECL chemiluminescence kit (Beyotime, Shanghai, China). Finally, the Gel-Pro Analyzer was used to analyze protein densitometry. The relative expression levels of all protein were normalized to β-actin.

The treated keratinocytes were lyzed with RAPI buffer (New Cell & Molecular Biotech, WB3100, China) containing proteinase and phosphatase inhibitor cocktail (Beyotime Biotechnology, P1045, China). Protein of each sample was separated by 10% SDS-PAGE (Biotides Biotech, WB2102, China) and transferred to polyvinylidene fluoride membranes (EMD Millipore, ISEQ00010, United States). The primary antibodies for western blotting included anti-STAT1 (1:1,000, CST, 14995, United States), anti-phospho-STAT1 (Tyr701) (1:1,000, CST, 9167, United States) and anti-IL-28R antibody (1:750, Abcam, ab224395, United Kingdom). The blots were then incubated with anti-rabbit IgG, HRP-linked antibody (1:3,000, CST, 7074, United States). The specific bands were observed by Tanon Imaging System (Tanon, China) with Immobilon Western Chemiluminescent HRP Substrate (Millipore, WBKLS0500, United States). The band intensity was calculated by ImageJ software (National Institutes of Health, Bethesda, MD) compared with GAPDH (1:10,000, Proteintech, 60004-1-lg, United States). The secondary antibody for GAPDH was Goat Anti-Mouse IgG H&L (HRP) (1:9,000, Abcam, ab6789, United Kingdom).