Mouse anti his c ab

High-five cells (1 × 10⁶/well) cultured as monolayers in serum free medium in 6-well titration plates were infected with the recombinant baculovirus constructs (AcBac-sccaIL-12, AcBac-sccaIL-12opt, AcBacΔCC-sccaIL-12, or AcBacΔCC-sccaIL-12opt) at MOI of 2, 5 or 10. To assess secreted recombinant protein, the cell culture SN were collected at 24, 48 and 72 h post infection, floating cells were spun down at 500×g for 5 min, and the cell-free SN were stored at −20 °C until use. Recombinant canine IL-12 in the cell culture SN was evaluated by dot-blot assay, using mouse anti-histidine C-terminal antibodies (mouse anti-His-C Ab, Invitrogen) as described below.

For dot-blot assays, nitrocellulose membranes placed in a blotter device (BioRad, Hercules, United States) were directly coated with proteins from cell culture SN at 150 µL/well. The membranes were blocked with tris-buffered saline, pH 7.6, containing 5 % powdered non-fat milk and 0.05 % Tween 20 and developed with mouse anti-His-C Ab (Invitrogen) diluted 1:5.000, goat anti-mouse IgG alkaline phosphatase-conjugate diluted 1:500 (Sigma-Aldrich), and alkaline phosphatase substrate (5-bromo-4-chloro-3-indolyl-phosphate and nitro blue tetrazolium, Sigma Aldrich). For Western blot analysis, samples of SN or cell lysate from similar volumes of cultures of High-five cells infected with AcBac-pFast-cont, AcBacΔCC-pFast-cont, AcBac-sccaIL-12, AcBacΔCC-sccaIL-12, AcBac-sccaIL-12opt or AcBacΔCC-sccaIL-12opt baculovirus contructs at a MOI 5 for 48 h were fractioned by polyacrylamide gel electrophoresis with dodecyl sodium sulfate (SDS–PAGE) and then transferred to nitrocellulose membranes (BioRad). Detection of his-tagged protein was performed as described in the dot-blot assay.