Ecl luminol reagent

After 6 rounds of plaqued selection, 9 selected vaccinia VG9/(SST-14)₂-HSA were propagated in BSC-40 cells. Then, the infected cells and supernatant were harvested and lysed through 3 freeze-thaw cycles. The lysates of vaccinia were separated by 10% SDS-PAGE gel and transferred to a PVDF membrane. The membrane was blocked with 3% BSA and incubated with primary antibodies against HSA (ab83465, Abcam, London, UK), somatostatin (ab53165, Abcam) and β-actin (Santa Cruz, CA, USA). Finally, corresponding HRP-conjugated anti-IgG secondary antibodies were incubated and the blots were developed by an ECL luminol reagent (Santa Cruz).

10 strains of vaccinia VG9-CD purified by plaqued selection were propagated in BSC-40 cells. Cells and supernatants were harvested and lysed by 3 cycles of freezing and thawing. Vaccinia lysate were separated by 10% SDS-PAGE gel and transferred to a PVDF membrane (Life Technologies, NY, USA). The membrane was blocked with non-fat milk and incubated with anti-yeast CD polyclonal antibodies (2485–4906, Bio-rad, CA, USA) overnight at 4 °C. Finally, blots were developed with HRP-conjugated anti-goat IgG and detected by an ECL luminol reagent (Santa Cruz, CA, USA).

The colon tissues were homogenized and lysed in RIPA buffer (Solarbio) containing PMSF (100 : 1) for 15 min. After centrifugation (4°C, 15000 rpm, 10 min), the supernatant was collected, and the protein concentration was quantified using BCA protein assay reagent (Beyotime). The protein lysates (50 μg protein) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Immobilon). After being blocked with 5% bovine serum albumin (BSA) solution, the membranes were incubated with primary antibodies against TNF-α, NF-κB p65, IκBα, and phosphorylated IκBα, followed by the appropriate secondary antibodies. Then, the protein-antibody complexes were developed with ECL Luminol reagent (Santa Cruz Biotechnology) and visualized.