Precast glgel tris glycine page

For relative expression quantification, infected cells were lysed in radioimmunoprecipitation assay lysis buffer (strong) (Cowin Biosciences, CW2333S) supplemented with 100× PMSF (Beyotime, ST505) and 100× ProteinSafe Protease Inhibitor Cocktail (TransGen, DI111-01). The protein lysate was centrifuged to remove debris. The supernatant was mixed with 5× Protein Loading Dye (Sangon Biotech, C508320-0001) and heated at 96° to 100°C for 10 min. Then, the mixed sample was centrifuged at 13,000 rpm for 20 min at 4°C. The sample was separated by 10% Precast-Glgel Tris-Glycine PAGE (Sangon Biotech, C651101-0001) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, IPVH00010). After probing with the respective antibodies, the PVDF membranes were finally analyzed with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, 34095).

Four cytokines (IL-1β, TNF-α, IL-17 and IL-6), four DEPs (BPGM, GPD1, APOE and APOC₂) were identified by the integrated Omic analysis, and five key proteins (LRP5, DVL1, β-catenin, Cacybp, Skp1) involved in the Wnt/β-catenin signaling pathway were further validated by Western blot analysis. Briefly, total proteins were extracted using Cell lysis buffer (PMSF 1:100) for Western and IP (Beyotime, China). The total protein concentrations were quantified with a BCA assay (Bio-Rad, Hercules, CA). Next, the target proteins were separated by 8–20% Precast-Glgel Tris-Glycine PAGE (Sangon, Biotech), transferred to PVDF membrane. Then the PVDF membrane was blocked for 1 h. Next, the PVDF membrane was incubated with β-actin, IL-1β, TNF-α, IL-17, IL-6, BPGM, GPD1, APOE, APOC, LRP5, DVL1, β-catenin, GSK-3β, Cacybp and Skp1 overnight at 4 °C. Subsequently, the PVDF membrane was washed three times in TBST and incubated with either secondary antibody goat anti-mouse IgG-HRP (diluted 1: 2000, absin, abs20001, China) or goat anti-rabbit IgG-HRP (diluted 1: 2000, absin, abs20002, China) for 1 h. Protein bands were detected with the help of a FluorChem Q scanner (Proteinsimple, CA, USA).