Ecl western blot detection reagents

The RAW264.7 cells were loaded into 6-well plates (2 × 10⁵ cells/well) and incubated for 24 h. After that, the original media were changed to new media with different concentrations of Se-POP-21 solution (0, 200, 400, or 800 μg/mL) or LPS solution (1 μg/mL) and incubated for 24 h continuously. Then, the supernatant was removed, and the cells were washed with iced PBS once. After that, 200 μL of RIPA lysate was added to each well, and the cell suspension was collected. After lysis on ice for 10 min, the cells were centrifuged at 4 °C at 12,000 RPM for 15 min, and the supernatant was collected. The protein concentration was determined using the BCA protein quantification kit. The proteins were separated by polyacrylamide gel electrophoresis (SDS–PAGE), transferred to a nitrocellulose (NC) filter membrane, and sealed with 5% skimmed milk powder prepared with PBST for 90 min. The primary antibody was diluted proportionally (anti-NF-κB 1:1000; anti-β-actin 1:5000) and incubated with the membrane at room temperature for 90 min. The HRP-conjugated secondary antibody was diluted (1:5000) and incubated with the membrane at room temperature for 90 min. The target proteins were detected using ECL Western blot detection reagents (Cwbiotech, Beijing, China).

Vero cells were infected with rSG10, rSG10-K1756A, or rSG10-G1780A and harvested at indicated time points. Protein samples were prepared using the ProteinExt_® Mammalian Total Protein Extraction Kit (Transgen, Beijing, China), separated by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Amersham Biosciences, Freiburg, Germany). Membrane was blocked with QuickBlock™ Western (Beyotime, Beijing, China) for 30 min and subsequently incubated with primary antibody at 4°C overnight. After washing three times with TBST, the membranes were incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies (Bioss Biotechnology; 1:10,000 dilution). Protein bands were then detected using ECL western blot detection reagents (CWBIO, Beijing, China).