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Figure 2
SapS is produced by S. aureus both in vitro and in infected macrophages. (A) Cultures were grown up to exponential phase and supernatants from SA564 ?sapS + pLI50_SapS-Spot and SA564 + pLI50_SecA-Spot (negative secretion control) were filtered and immunoprecipitated using anti-Spot magnetic beads, while bacterial pellets were resuspended and lysed in PBS with lysostaphin, a protease inhibitor cocktail, DNAase I and fractionated. Cytoplasm (C), membrane (M), cell wall (W), and immunoprecipitated proteins (IP) from supernatants were migrated on SDS-PAGEs, transferred to PVDF membranes for Western-blot analyses using monoclonal mouse anti-Spot antibody as primary antibody (Chromotek, Germany) and an HRP-coupled donkey-anti-mouse antibody as secondary antibody (Jackson Immuno Research). Data are representative of three different experiments. (B) RAW 264.7 macrophages (2.5 × 105 cells/well) were infected with S. aureus at a MOI of 20, and non-phagocytosed bacteria were subsequently removed by gentamicin/lysostaphin treatment. Infected macrophages were lysed in 0.1% Triton X-100 and centrifuged at 14,000× g. The macrophage lysates were immunoprecipitated with anti-Spot magnetic beads (Chromotek), while the pellets of the intracellular bacteria were resuspended in an equal volume of lysis buffer. Immunoprecipitated proteins (IP) and bacterial pellets (BP) were separated on SDS-PAGE and observed with an anti-Spot antibody as described in (A). Data are representative of three different experiments (M kDa: molecular markers).