Fig 4.

SMAMPs interact with LTA. (A and B) RAW 264.7 cells (10⁶ per ml) (open bars) were pretreated with SMAMPs (1.0 μg/ml) or DMSO (control). The cells were then washed and incubated in antibiotic-free RPMI for 1 h before stimulation with S. aureus (MOI = 10) for 14 h. The cells (solid bars) were also pretreated with SMAMPs and stimulated with S. aureus (MOI = 10) for 14 h without medium change. The supernatants were assessed for TNF and IL-6 production by ELISA. The data shown represent the averages and SEM of triplicate determinations and are representative of three independent experiments. (C to F) UV titration of 1% DMSO (C and E) and 100 μg/ml SMAMP 02 (D and F) with increasing concentrations of LTA (C and D) or Pam₃CSK₄ (E and F). The results are representative of three independent determinations. (G) TLR2 binding competition assay. Wells were coated with LTA or Pam₃CSK₄ (0.5 μg/well) in chloroform-ethanol (1:9), followed by incubation with SMAMP (1 μg/ml) and TLR2-Fc (0.5 μg/ml). Alkaline phosphatase activity was determined using BluePhos phosphatase substrate. The data shown represent the averages and SEM of triplicate determinations and are representative of three independent experiments.