Figure 5

Effects of PBEF knock down on IL-8 protein & mRNA expression in A549 cells (A-D) and in lung primary small airway epithelial cells (E-F). A. Representative western blotting image of secreted IL-8 protein detections. After starving for serum overnight, A549 cells were transfected with the vehicle control (C), scrambled siRNA (Sc), or PBEF stealth siRNA (Si) for 48 h before treatment without or with TNFα (15 ng/ml) for 24 hours. B. Quantitation of secreted IL-8 protein levels by densitometric analysis. **p < 0.01 vs. control. C. Representative RT-PCR gel image of IL-8 and β-actin mRNA detections. A549 cells were grown and transfected with the PBEF siRNA and other controls as described above before treatment without or with TNFα (15 ng/ml) for 4 hours. Total cell RNA was reverse-transcribed, amplified by PCR using gene specific primers (Table 1), separated by 1.5% agarose electrophoresis and visualized with ethidium bromide. D. Quantitation of IL-8 mRNA level by densitometric analysis. **p < 0.01 vs. control. E. Representative western blotting images of cell lysate PBEF and secreted IL-8 protein detections. Lung primary small airway epithelial cells were transfected with control (C), PBEF stealth siRNA (Si), or scrambled RNA (Sc) for 48 h before treatment without or with TNFα (10 ng/ml) for 12 hours. PBEF in cell lysate and secreted IL-8 protein were immunodetected as described in Figures 1 and 2. F. Quantitation of IL-8 and PBEF protein levels by densitometric analysis. **p < 0.01 vs. control.