FIG 7.

Cysteine protease inhibition results in a decrease in mRNA expression of TNF-α and IL-1β but does not have an effect on protein secretion levels for all three proinflammatory cytokines examined. ST-7 (B) lysates were pretreated with 100 μM cysteine protease inhibitor, E-64, for 1 hour before coincubation with RAW264.7 cells at 37°C. (A) Representative Western blots for activation of MAPKs. Cells were lysed, and cell extracts were analyzed by Western blotting using phospho-ERK, total ERK, phospho-JNK, total JNK, phospho-p38, and p38 antibodies. (B) mRNA levels of TNF-α, IL-1β, IL-6, and GAPDH were measured by qRT-PCR. The values are expressed as mean relative transcript levels normalized to GAPDH and are expressed relative to the 0-h time point, which is set at 1. (C) Concentrations of IL-6, TNF-α, and IL-1β in the supernatants were measured by ELISA. Each value represents the mean and standard deviation from at least 3 individual experiments. *, P < 0.05; ***, P < 0.005 (representing a statistically significant difference between inhibitor and vehicle treatments).