Extracellular Signal-Regulated Kinase Is a Direct Target of the Anti-Inflammatory Compound Amentoflavone Derived from Torreya nucifera
Figure 4
Effect of amentoflavone on signaling factors upstream of AP-1. (a) Phosphorylated and total protein levels of ERK, p38, JNK, and β-actin in RAW264.7 cell lysates were determined by immunoblotting analyses using phosphospecific or total protein antibodies. Relative intensities were calculated by densitometric scanning. (b) HEK293 cells cotransfected with an AP-1-Luc plasmid construct (1 μg/mL) and β-gal (as a transfection control) were treated with U0126 (U0, 20 μM), SB203580 (SB, 20 μM), or SP600125 (SP, 20 μM) in the presence or absence of PMA (100 nM). Luciferase activity was measured using a luminometer. (c) The kinase activities of immunoprecipitated ERK prepared from LPS-treated RAW264.7 cells and purified ERK (ERK1 and ERK2) were determined in a direct kinase assay using purified enzymes or by measuring the level of phospho-MBP. The control (set as 100%) was the activity of each enzyme (ERK1 or ERK2) obtained after treatment with vehicle. The level of phosphorylated MBP was measured by immunoblotting analysis. (d) The total level of c-Fos in the nuclear fraction of LPS-treated RAW264.7 cells after U0, SB, or SP treatment was determined by immunoblotting analyses using specific antibodies. (e) An interaction between ERK and c-Fos was evaluated by immunoprecipitation and immunoblotting analyses. RAW264.7 cells (5 × 106 cells/mL) were incubated with amentoflavone (200 μM) in the presence or absence of LPS (1 μg/mL) for 30 min. c-Fos was immunoprecipitated from whole cell lysates using a specific antibody, followed by immunoblotting with antibodies to ERK, c-Fos, and rabbit immunoglobulin heavy chain. (f) Putative anti-inflammatory signaling pathway induced by amentoflavone treatment. compared to the control.
