Protium javanicum Burm. Methanol Extract Attenuates LPS-Induced Inflammatory Activities in Macrophage-Like RAW264.7 Cells
Figure 3
Effect of Pj-ME on the NF-κB and its upstream signaling cascade in LPS-stimulated RAW264.7 cells. ((a) left panel, (a) right panel and (c)) Western blot analysis was performed to detect protein expression levels in whole cell lysates or nuclear extracts from RAW264.7 cells treated with Pj-ME (200 μg/ml) for 30 min followed by LPS exposure (1 μg/ml) over various lengths of incubation times. Levels of phosphorylated and total p85, IKKα/β, IκBα, p50, and p65 at 5, 15, 30, and 60 min, and Syk and Src levels at 2, 3, and 5 min were determined. β-Actin was used as a loading control. (b) HEK293 cells cotransfected with NF-κB-Luc (1 μg/ml) and β-gal (as transfection control) plasmid constructs were treated with Pj-ME in the presence or absence of the adaptor molecule MyD88 (1 μg/ml). Luciferase activity was measured by using luminescence. ((d) and (e)) Inhibitory activity of Pj-ME (100 and 200 μg/ml) on autophosphorylation of Syk and Src overexpressed in HEK293 cells was determined by Western blot analysis with antibodies specific to phospho-Src or phospho-Syk. Data (b) expressed as mean± SD are representative of 3 independent experiments. ##p< 0.01 with respect to untreated group and p< 0.01 with respect to treated group.