FIG 3.

Inhibition of ERK leads to reduced cytokine expression in response to Blastocystis. RAW264.7 cells were pretreated with either 100 μM PD98059 or ethanol vehicle dissolved in RPMI complete medium for 1 hour prior to stimulation with Blastocysis ST-7 at an MOI of 10 for the indicated times. (A) Representative Western blots for MAPK activation in RAW264.7 cells. The cells were lysed and processed by SDS-PAGE. Membranes were then blotted with phospho-ERK, ERK, phospho-JNK, JNK, phospho-p38, and p38 antibodies. Each blot is representative of three separate experiments. (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 culture supernatants were measured by ELISA. Each value represents the mean and standard deviation from at least 3 individual experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.005 (representing a statistically significant difference between inhibitor and vehicle treatments).