IRAK1/4-Targeted Anti-Inflammatory Action of Caffeic Acid
Figure 4
Effect of CA on the activation of AP-1 upstream signaling. (a) Levels of total or phosphoforms of MAPK (JNK, p38, and ERK) and JNK upstream enzymes (MKK4/7, TAK1, IRAK4, and IRAK1) from total lysates were determined by immunoblotting analyses with specific antibodies. (b) IRAK1 and IRAK4 kinase activities were determined by direct kinase assays with purified enzymes. The control for each enzyme (IRAK1 or IRAK4) was the activity obtained with vehicle treatment alone and was set at 100%. and , compared to the control. (c) RAW264.7 cells (5 × 106 cells/mL) were incubated with CA (400 μM) in the presence or absence of LPS (1 μg/mL) for 5 min. After preparing total lysates, the level of TAK1 binding to IRAK4 was identified by immunoprecipitation with an IRAK4 antibody and immunoblotting with antibodies against the rabbit immunoglobulin heavy chain and TAK1. (d) Culture supernatants prepared from LPS-treated RAW264.7 cells that were pretreated with a standard JNK inhibitor (SP600125 (SP)) were assayed for NO and PGE2. HC: heavy chain; , compared to the control.
