Horseradish peroxidase conjugated anti mouse antibody

Changes in the levels of intracellular viral proteins sequential expression were analyzed by western blot as previously described^{30 (link)}. Briefly, infected Vero cells were lysed in RIPA buffer (1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 1 mM CaCl₂, 0.5 mM MgCl₂, and protease inhibitor cocktail) after 4, 8, 12, and 24 h.p.i., the insoluble debris was removed by centrifugation at 16,000 g. Forty μg of whole-cell extracts were resolved on 12% SDS-PAGE and transferred to polyvinylidene difluoride membranes (PVDF, Thermo Scientific, IL, USA). The membranes were incubated with anti-PIXV mouse primary polyclonal antibody (1/1000) overnight at 4 °C and then with a secondary horseradish peroxidase-conjugated anti-mouse antibody (1/800) (Promega Corporation, Madison, WI) for 1 h at RT. Anti-GAPDH antibody was used as a loading control (Sigma-Aldrich, St. Louis, MO, 1:5000). Blots were developed using a chemiluminiscence detection kit (ECL; GE Amersham) for the visualization of reactive bands.

Protein extraction and immunoblotting were performed as described previously [22 (link)]. Briefly, 1 × 10⁶ cells were lysed in 100 μL M-PER lysis buffer containing Halt protease and a phosphatase inhibitor cocktail (Pierce Biotechnology, Rockford, IL, USA). Cell lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride microporous membrane (Immobilon-P Transfer Membrane, Millipore, Bedford, MA, USA) using the NuPAGE system (Life Technologies). The membranes were blocked with Block Ace (DS Pharma Biomedical, Osaka, Japan) and probed with the antibodies to LANA-1 [23 (link)], RTA [21 (link)], vIL-6 [20 (link)], ORF59 [24 (link)], or Lyn (H-6, sc-7274, Santa Cruz Biotechnology), followed by a horseradish peroxidase-conjugated anti-mouse antibody (Promega, Madison, WI, USA) with an immunoreaction enhancer solution (Can Get Signal, Toyobo, Osaka, Japan). Blots were visualized using Super-Signal West Femto Chemiluminescent Substrate (Thermo Fisher Scientific).