Clarity western peroxide reagent

MARC-145 cells seeded in six-well plates were infected with PRRSV. At 24 hpi, the cells were harvested with 1 × SDS loading buffer (Beyotime) and boiled for 10 min. After centrifuging at 12,000× g, 4 °C for 10 min, proteins were separated by electrophoresis on 12% polyacrylamide gels (EpiZyme Biotechnology, Shanghai, China) and electro-blotted onto polyvinylidene difluoride membranes (Millipore, MA, USA). Next, the membranes were blocked at room temperature with 5% BSA for 3 h and incubated with the antibody specific to PRRSV-N protein, followed by incubation with horseradish peroxidase-conjugated Goat Anti-Mouse IgG (Beyotime). Finally, the resultant images were visualized and collected using Clarity Western Peroxide Reagent (Bio-Rad, CA, USA).

Frozen MP, cell, and MS serum samples were thawed, and 0.5 μL of each of the samples was loaded onto nitrocellulose membranes and left to dry overnight. Ponceau S staining was used for protein loading standardization. A measure of 5% milk and TBST were used to block (2 h) and rinse the blots (3X) after staining. Membranes were immunoblotted for cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS), and 3-mercaptopyruvate sulfur transferase (MST) and incubated in Clarity Western Peroxide Reagent and visualized using Clarity Western Luminol/Enhancer Reagent (Biorad, Hercules, CA, USA). All primary antibodies were used at 1:500 dilution and incubated overnight. Secondary antibodies were used at 1:2000 dilution and incubated for 1 h at room temperature before washing and ECL reactions.
ChemiDoc imaging system (Biorad, Hercules, CA, USA) was used to develop images of membranes. Densitometry was performed using ImageJ analysis software, v.153 (NIH, Bethesda, MD, USA). Data were normalized to total protein using Ponceau S Staining (Sigma biochemicals, St. Louis, MO, USA).