Macrophage Signaling Pathway Activation

B10R macrophages were infected for 0.5 h, 1 h, and 3 h, with similar numbers of viral particles as in footpad injection, or stimulated with LPS (100 ng/ml) for 60 minutes as performed for induction of NF-κB nuclear translocation; uninfected wells served as negative controls. Experiments were performed in triplicate and proteins were extracted as previously described [15 (link)]. Briefly, following cell lysis and protein extraction of infected and uninfected cells, protein contents were dosed by Bradford Assay (Bio-Rad, Mississauga, ON, Canada). Proteins were resuspended in an SDS sample buffer containing bromophenol blue and beta-mercapto-ethanol, separated by SDS-PAGE using 10% acrylamide gels, and transferred to PVDF membranes (Perkin Elmer, Waltham, MA). Membranes were blocked for 1 hour in TBS- 0.05% Tween 20 containing 5% BSA before overnight incubation with the primary antibody (e.g. phospho-p44/42 (ERK 1/2), ERK 1/2, phospho-p38, p38, phospho-IRF3, IRF3, phospho-TBK1/NAK, TBK). Membranes were washed and incubated with the rabbit secondary anti-HRP-conjugated antibody (GE Healthcare, Baie d’Urfe, QC, Canada), and proteins were visualized by ECL Western Blot Detection System (GE Healthcare). USA).