Publication protocol
"Sf-RVN and Sf9 cells in ESF 921 were seeded into six-well plates at densities of 1 x 106 cells/well. The cells were then mock-infected with ESF 921 or infected with Sf-rhabdovirus-negative stocks of BacPAK6-ΔChi/Cath, AcP(−)p6.9hSEAP, or AcP(−)p6.9hEPO at multiplicities of infection (MOIs) of either 0.1 or 5 plaque-forming units (pfu)/cell. At various times post infection, the infected cells were harvested, cell densities were measured, and the cells were pelleted by low speed centrifugation. The cells and cell-free media were then processed in various ways, depending upon the nature of the model protein being expressed and purpose of the experiment, as described below. In each case, however, the levels of recombinant protein in cell extracts and/or cell-free media were measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting [18, 19] with protein- or tag-specific primary antibodies and alkaline phosphatase-conjugated secondary antibodies, as specified below. Immunoreactive proteins were visualized using a standard alkaline phosphatase-based color reaction [20] and relative intensities were estimated by scanning and quantitating the bands using Image J software.
For β-gal, infected cell pellets were used to prepare cytoplasmic extracts for enzyme activity assays, as described previously [21]. Immunoblotting was performed using rabbit anti-β-gal (EMD Millipore Corporation, Germany) and alkaline phosphatase conjugated goat anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO) as the primary and secondary probes, respectively."
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