Protein loading buffer

RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific) was applied for protein extraction from cells and tissues. The lysates were then mixed with protein loading buffer (Beyotime) and denatured in the boiling water bath for 10 min. The protein products were immediately used for the expression determination as previously described [29 (link)]. Antibodies were purchased from Cell Signaling Technology (CST, Boston, MA, USA): phosphorylated (Ser 9) glycogen synthase kinase 3 beta (p-GSK3β; #5558, 1:1000), GSK3β (#12456, 1:1000), β-catenin (#8480, 1:1000), E-cadherin (#3195, 1:1000), N-cadherin (#13116, 1:1000), reference gene GAPDH (#5174, 1:1000), and Anti-rabbit IgG, HRP-linked second antibody (#7074, 1:3000). Protein blotting could be emerged using SignalFire™ Elite ECL Reagent (CST) and protein quantification was performed by ImageLab software version 4.1 (Bio-Rad).

Chicken embryo fibroblasts was inoculated with viruses (MOI = 0.01) and incubated for 1 h at 37 °C. Cells were washed twice with phosphate buffered saline (PBS, pH 7.2). Thereafter, M199 medium containing 1% FBS was added and incubated for 12 h. Cells were washed once with pre-cooled PBS (4 °C) and lysed with 200 μL of RIPA Lysis Buffer (strong) (CWBIO, Beijing, China) individually on ice for 15–20 min. Supernatants were collected by centrifugation at 12000 r/min for 10 min at 4 °C and mixed with protein loading buffer (Beyotime Biotechnology, China). Following boiling at 100 °C for 6–8 min, samples were subjected to 12% SDS-PAGE, and transferred to a PVDF membrane. The membrane which was first blocked in TBST containing 5% non-fat powdered milk at 25 °C for 1 h was incubated with the primary antibody against the H5 14th peptide (diluted to 1:1000 with TBST), and then incubated with the secondary antibody (Goat Anti-Chicken IgY (H + L) HRP, Abcam, USA, diluted 1:5000 with TBST). Meanwhile, protein bands of β-actin were incubated successively with the primary antibody (Anti-β-actin monoclonal antibody, Sigma Company, USA) and secondary antibody (HRP labelled anti-mouse IgG goat polyclonal antibody, Abcam, USA). Protein bands were developed using a chemiluminescence imaging analysis system (Tanon 5200, Tanon Biotech, China).

The experiment consisted of 5 groups, including the control group, LPS group (1 μg/mL LPS), low-dose group (1 μg/mL LPS + 1.25 mg/mL XXMD-C), medium-dose group (1 μg/mL LPS + 2.5 mg/mL XXMD-C), and high-dose group (1 μg/mL LPS + 5 mg/mL XXMD-C). BV-2 cells were cultured with or without XXMD-C and induced with LPS for 24 h. After washing twice with pre-cooled PBS, the cells were lysed with 150 μL RIPA lysis buffer (G2002, servicebio, China) on ice for 30 min. The lysate was collected in a 1.5 mL EP tube using a clean cell scraper and centrifuged at 13,400 g at 4°C to obtain the supernatant, and the protein concentration of each group was determined according to the instructions of the BCA kit. Proteins were denatured by adding protein loading buffer (070121211027, Beyotime, China) and boiling for 5 min at 100°C. The proteins were then separated for subsequent gel electrophoresis. The samples were subjected to 10% SDS-PAGE electrophoresis, and then transferred onto a polyvinylidene fluoride (PVDF) membrane in ice bath. The PVDF membrane was blocked with 5% skimmed milk at room temperature for 1.5 h and incubated overnight in the primary antibody solution. Finally, the PVDF membrane was incubated in the secondary antibody solution for 1 h. After washing, the protein bands were visualized using ECL solution for 1–2 min and analyzed for the gray value.