Figure 4

Staphylococcal LTA inhibits LPS-induced B cell proliferation by decreasing ERK phosphorylation. (a) Splenocytes or purified B cells (2.5 × 10⁷ cells/ml) were stimulated with LPS (0.1 μg/ml) and/or Sa.LTA (50 μg/ml) for 24 h. After stimulation, the cells were lysed and equal amounts of proteins were subjected to Western blot analysis using antibodies specific to the non-phosphorylated or phosphorylated form of ERK. (b) CFSE-labeled splenocytes (1 × 10⁶ cells/ml) were pre-treated with an ERK-specific inhibitor, U0126 (5 or 10 μM), for 1 h and subsequently stimulated with LPS (0.1 μg/ml) for 72 h. After stimulation, the cells were stained with PerCP-conjugated rat anti-mouse CD45R/B220 antibody and B cell proliferation was determined by flow cytometry. (c) Splenocytes (2.5 × 10⁷ cells/ml) were pre-treated with a JAK inhibitor (0.1 or 0.5 μM) for 1 h and subsequently stimulated with Sa.LTA (50 μg/ml) and/or LPS (0.1 μg/ml) for 24 h. After stimulation, the cells were lysed and equal amounts of proteins were subjected to Western blot analysis using antibodies specific to the non-phosphorylated or phosphorylated forms of ERK. The gels were run under the same experimental conditions and the blots were representative of three independent experiments. The results were shown as cropped blots (Whole blots are shown in Supplementary Figure S1). Relative p-ERK values were obtained by the densitometric analysis of Western blot data from three independent experiments.