Effect of IRAK4 kinase activity on the activation of NF-κB. (1) VSMCs were pretreated with 1 µM IRAK1/4 inhibitor for 1 h, followed by 10 µg/mL LPS stimulation for 2 h. The cell lysates were analyzed by western blot analysis against anti-IRAK4, anti-p-IRAK4, anti-NF-κB p65, and anti-p-NF-κB p65 (A and B). Data represents the mean ± SEM of triplicate samples from a single experiment, and the results are representative of three independent experiments. ***P < 0.001 compared with the control group; #P < 0.05 compared with the LPS group; ##P < 0.01 compared with the LPS group; ###P < 0.001 compared with the LPS group. (2) VSMCs were untransfected or transfected with siR-IRAK4 for 48 h, and then stimulated by LPS (10 µg/mL) for 2 h. Total RNA was subjected to RT-PCR to measure the relative expression of IRAK4, NF-κB p65 (C and D). The expression of IRAK4 and NF-κB p65 normalized to β-actin expressionis demonstrated. Data represents the mean ± SEM of triplicate samples from a single experiment, and the results are representative of three independent experiments. *P < 0.05 compared with the control group; ***P < 0.001 compared with the control group; #P < 0.05 compared with the LPS group; ##P < 0.01 compared with the LPS group; ###P < 0.001 compared with the LPS group; &&&P < 0.001 compared with the LPS + siR-IRAK4 group.