FIG 2.

SAP binds B. anthracis PGN in a calcium-dependent manner and enhances monocyte TNF-α production in response to PGN. Human serum was pretreated with EGTA or not pretreated and then incubated with PGN for 1 h at 4°C. PGN was then pelleted by centrifugation and washed. Bound proteins were eluted in LDS sample buffer by heating. (A) Samples were then subjected to PAGE and immunoblotting for SAP. (B) Membranes were then stripped and analyzed for IgG via Western blotting. Images are representative of 3 human donor sera. Subsequently, PBMCs were left unstimulated or were stimulated with PGN (10 μg/ml) in the presence of 1% FCS or 1% FCS supplemented with recombinant SAP (1, 5, 10, or 40 μg/ml) (C) or IgG (50, 100, 200, or 400 μg/ml) (D) for 12 h. Cells were then harvested and stained for CD14 and TNF-α. Flow cytometry was utilized to assess the percentage of CD14 cells that produced TNF-α. Graphs show means ± SD from 3 independent donors.