FIGURE 1.

Pretreatment with MDP inhibits proinflammatory responses to subsequent MDP treatment. A and C, RAW264.7 cells were pretreated with MDP (100 μg/ml) for 4 h, washed, and restimulated with MDP (100 μg/ml), LPS (10 ng/ml), CpG-B (1 μm), Pam3CSK4 (10 μg/ml), or flagellin (3 μg/ml) for 24 h. B, BMDM were stimulated with LPS (0.5 ng/ml) for 6 h and then incubated with MDP (100 μg/ml) for 4 h in the absence of LPS. After several washes, the cells were restimulated with MDP (100 μg/ml) for 24 h. Supernatants were subjected to ELISA for TNF-α. D and E, wild-type BMDM or Rip2-deficient BMDM were pretreated with LPS (0.1 ng/ml) for 6 h and stimulated with MDP for 4 h in the absence of LPS. The cells were washed and restimulated with MDP for 30 min. D, cell extracts were subjected to Western blot analysis for IκBα, p-ERK, p-JNK, and actin. E, cells were fixed and permeabilized for 5 min. Immunofluorescent staining for p65 was performed using anti-p65 Ab followed by Alexa Fluor 594 detection antibody. Cells were analyzed under a fluorescence microscope. Error bars represent the mean ± S.D. of triplicates. **, p < 0.005. Data are representative of three independent experiments with similar results. n.s., nonspecific; N.D., not detected.