Figure 3.

Surface TLR activation antagonize TLR8-IRF5 signaling and triggers a rapid loss of IRAK-1 detection on Western blot. (A) Levels of nuclear IRF5 in monocytes stimulated for 2 h with TLR8-agonist CL075 (1 μg/ml), TLR4-agonist LPS (1 μg/ml), ECO 17-1 (1 × 10⁶/ml), or TLR5-agonist Flagellin (FLA, 1 μg/ml) or combinations of these. Quantification of nuclear IRF5 was done by IF and scanR analysis (mean + SEM, N = 4).*indicate significant difference from CL075, while ^#indicate significant difference from no stimuli. ^##p < 0.01, ^###p < 0.001, **p < 0.01, and ***p < 0.001. (B) Immunoblots of total IRAK-1, IRAK-2, IRAK-4, and IRF3-P after 30 min of treatment with CL075 (1 μg/ml), LPS (1 μg/ml), or ECO 17-1 (1 × 10⁶/ml) alone and in combinations. GAPDH served as a loading control, and a representative of four independent experiments is shown. (C) Immunoblots of total IRAK-1, IRAK-2, and IRAK-4 after 30 min of treatment with FLS-1 (100 ng/ml), CL075 (1 μg/ml), or live GBS (5 × 10⁶/ml) alone and in combinations. A representative of four independent experiments is shown. (D) Kinetics of IRF5 and p65/RelA nuclear accumulation in monocytes after treatment with CL075 (1 μg/ml) alone or combined with FLS-1 (100 ng/ml). Quantification of IRF5 and p65/RelA positively stained nuclei was done by IF and scanR analysis. A representative of four independent experiments is shown. (E) Effects of TLR stimulation for 15 and 30 min on IRAK-1 detection on immunoblot. Cells were untreated (NS) or stimulated with FSL-1 (100 ng/ml), LPS (1 μg/ml), and FLA (1 μg/ml). A representative experiment out of three independent experiments is shown.