Fig 3.

SMAMP-mediated cytokine inhibition is specific for LTA. (A) RAW 264.7 cells (10⁶ per ml) were stimulated with LPS (100 ng/ml), poly(I · C) (1.0 μg/ml), R848 (3.1 μg/ml), and CpG oligonucleotide and ODN (10.0 μg/ml) in the presence of three different SMAMPs (1.0 μg/ml) for 12 h. TNF production was measured in the stimulation supernatants by ELISA. RAW 264.7 cells (10⁶ per ml) were pretreated with SMAMPs or DMSO (control) for 1 h before stimulation. (B) HEK-hTLR2 cells (0.5 × 10⁶ per ml) were pretreated with 1.0 μg/ml SMAMPs or DMSO (control) for 30 min and stimulated with different TLR2 ligands: FSL-1 (10 ng/ml), Pam₂CSK₄ (10 ng/ml), Pam₃CSK₄ (100 ng/ml), PgLPS (1.0 μg/ml), LM-MS (100 ng/ml), and LTA (500 ng/ml). After 5 h of incubation, NF-κB-induced SEAP production was determined by alkaline phosphatase activity in the stimulation supernatants using Quanti-Blue as the substrate. The data shown represent the averages and SEM of triplicate determinations and are representative of three independent experiments.