Effects of LPS prestimulation on SOCS expression and on phosphorylation of NFκB-p65 and p38 after a second challenge with antigen, LPS, or a combination of both. BMMC were prestimulated with 10 ng/ml LPS or 100 ng/ml LPS for 18 hours. Next, the cells were stimulated with 20 ng/ml antigen, 1 μg/ml LPS, or a combination of both for 30 min. Figure 4A: NFκB-p65 phosphorylation was increased by co-stimulation with antigen + LPS and this synergistic effect was reduced by prestimulation with LPS, probably corresponding to the reduction in TNFα and IL-6 production. Under prestimulation, p38 phosphorylation was also reduced when stimulated with the combination of Ag and LPS. Figure 4B: Prestimulation with LPS by itself resulted in increased expression of SOCS-1 and -3 in MC/9 and SOCS-3 in BMMC cells. The blots shown were typical results from three experiments. For Figure 4B, images were cropped to allow direct visual comparison from non-stimulated and stimulated cells from the same gel. Fold induction is the mean of three individual experiments from two BMMC cultures.