Figure 4.

Luteolin directly inhibits LPS-induced IκB kinase activity in IEC-18 cells. (a) and (c), IEC-18 cells were pretreated with luteolin (50 µm) for 1 hr, and then stimulated with LPS (10 µg/ml) for 20 min (a) or infected with Ad5wtNIK (m.o.i 50) for 16 hr (c). Whole cell extract was immunoprecipitated with anti-IKKγ/protein-A beads and the kinase reaction was performed using GST-IκB (1–54) as a substrate as described under ‘Materials and methods’. Substrate protein was resolved by gel electrophoresis, and phosphate incorporation was assessed by autoradiography and PhosphorImager analysis. Coomassie Blue staining and Western blot for IKKβ show equal loading (2nd and 3rd blot, respectively). (b) IEC-18 cells were coinfected with Ad5κB-LUC and Ad5wtNIK in the presence or absence of luteolin (50 µm). Luciferase assay was measured using an Lmax microplate reader, and results were normalized for extract protein concentrations. (d) to evaluate whether luteolin inhibits IKK or IRAK-1 directly, immunoprecipitated IKK or IRAK-1 from LPS-stimulated IEC-18 cells were aliquoted into kinase reaction buffer in presence of various concentrations of luteolin or DMSO vehicle. IKK or IRAK-1 assay was performed as described in the Materials and Methods. These results are representative of at least two independent experiments. Veh, DMSO vehicle; Lut, Luteolin.