Figure 4.

Effect of AQCA on IRAK1 enzyme activity. (a,b) the inhibitory effect of AQCA (50 μM) or staurosporin (STS) (150 nM) on the enzyme activity of Src, Syk, IRAK1, IRAK4, or TAK1 was determined using a conventional kinase assay with purified enzymes provided from Millipore (Billerica, MA, USA). The control was set as 100% activity for each enzyme with vehicle treatment only. (c left panel) HEK293 cells transfected with IRAK1 (1 μg/mL) for 36 h were treated with AQCA (100 μM) for 12 h. The levels of total or phosphorylated p50, p65, p38, IRAK1, Flag, and β-actin in whole cell lysates were identified using immunoblot analyses. Relative intensity (c right panel) was calculated relative to β-actin levels using the DNR Bio-Imaging system. All data (a–c left panel) are expressed as the mean ± SD of experiments performed with three samples. * p < 0.05 and ** p < 0.01 compared to the control (purified enzyme alone or Flag-IRAK1 alone) groups.