Sqstm1 p62

Cell or tissue samples were separately harvested and lysed in RIPA buffer (CWbio, Beijing, China) containing 0.1 mg/mL PMSF (Keygen, Nanjing, China), protease inhibitor, and Phospho-stop (Roche, Mannheim, Germany). Protein aliquots (30 lg) were separated by 10% SDS-PAGE and transferred to 0.45-lm PVDF membranes (Millipore, Billerica, MA, USA). The blots were blocked for 1 h at room temperature and incubated separately with primary antibodies (diluted 1:1000) against ATG16L1, P-ATG16L1, LC3I/II, SQSTM1/P62 (Abclonal, Massachusetts, USA) (rabbit anti human), NOD2 (Novus, Missouri, USA) (mouse anti human), and GAPDH (Proteintech, Wuhan, China) (rabbit anti human) overnight at 4 °C. The PVDF membranes were washed with TBS–Tween 20 and then incubated separately with the appropriate HRP-conjugated secondary antibodies (diluted 1:5000) (CST, Massachusetts, USA) (goat anti-mouse, goat anti-rabbit) for 1 h at room temperature. After rinsing, the signal on the PVDF membrane was detected by the enhanced chemiluminescence method. The relative protein expression is presented as the ratio of target protein band intensity to GAPDH band intensity using ImageJ software (NIH, Bethesda, MD, USA).

The procedure was carried out as described in a previous study (Wang et al., 2020 (link)). In brief, 150 μl of lysis buffer (ThermoFisher Scientific, China) was used to lyse the cells. The resultant proteins were separated and then transferred to a polyvinylidene fluoride membrane. The membrane was, then, incubated for 1 h in 5% non-fat milk and incubated for 2 h with the following primary antibodies: anti-PEDV N-protein (Medgene Labs, United States), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; ABclonal, China), anti-microtubule-associated protein 1 light chain 3 alpha isoform IIB (LC3II; NOVUS, United States), and anti-sequestosome 1 (SQSTM1/P62; ABclonal, China), followed by three washes with PBST. The membranes were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse and anti-rabbit IgG (H+L) secondary antibodies (Bioworld, China). Immunoreactive protein bands were visualized using an ECL Kit (Millipore, China).

Ononin (99% purit, IO0040) was purchased from Solarbio Science & Technology. Dimethylsulfoxide (DMSO), LPS (E. coli: Serotype O55:B5), ATP, poly(dA:dT), and phorbol 12‐myristate 13‐acetate (PMA) were obtained from Sigma‐Aldrich. Muramyl dipeptide and flagellin (Salmonella typhimurium) were obtained from InvivoGen. DSS was obtained from MP Biomedicals Inc. The primary antibodies of IL‐1β, caspase‐1, LC3, SQSTM1/p62, TOM20, β‐actin, and HRP Goat IgG (H+L) were obtained from ABclonal. Diamidino‐phenyl‐indole (DAPI) was brought from Sigma‐Aldrich.