Figure 4

The effects of ISO-1 on IκB, p38 MAPK activation, NF-κB DNA binding, and IL-6 expression in V. vulnificus -infected PBMCs. Human PBMCs (2 × 10⁶/ml) were infected with V. vulnificus (Vv05191; MOI = 1) for 1 h and co-treated with ISO-1 (50 μM). Western blotting was used to measure IκB (Ser32) (A) and p38 MAPK (Thr180/Tyr182) (B) phosphorylation. β-actin was the internal control. Data are representative of three individual experiments. (C) Effects of ISO-1 on NF-κB binding. Nuclear proteins were extracted from cells after they had been infected with V. vulnificus and co-treated with or without ISO-1. An EMSA assay was used to analyse V. vulnificus-infected cells co-treated with ISO-1 for 15 min and to analyse nuclear proteins (3 μg). Lane 1: Control; Lane 2: V. vulnificus-infected cells; Lane 3: V. vulnificus-infected cells + ISO-1; Lane 4: ISO-1 only; (D) Effects of ISO-1 on IL-6 mRNA expression. Human PBMCs (2 × 10⁶/ml) were infected with V. vulnificus (Vv05191; MOI = 1) for 1 h and co-treated with ISO-1 (50 μM). The ratio represents the quantitative densitometry analysis of bands compared with untreated controls. β-actin was the internal control. For real-time RT-PCR analysis, the data is shown as a normalized ratio. Data are representative of three individual experiments. Data are the means ± SD from three individual experiments. *P < 0.05 vs. V. vulnificus infection only.