Figure 4

PBEF stealth siRNA-mediated silencing of PBEF protein and mRNA expression in A549 cells. A. Representative western blotting image of PBEF protein detections. After starving for serum overnight, A549 cells were transfected with the control (C), scrambled siRNA (Sc), or 50 nmolar PBEF stealth siRNA (Si) for 48 h before treatment without or with TNFα (15 ng/ml) for 24 hours. Cell lysate PBEF and β-actin protein were immunodetected as described. B. Quantitation of PBEF protein levels by densitometric analysis. A549 Cell lysate PBEF protein levels in various treatments were quantified by densitometric analysis. **p < 0.01. C. A representative RT-PCR gel image of PBEF and β-actin mRNA detections. A549 cells were grown and transfected with the PBEF siRNA and other controls as described above and then treated 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. Quantification of PBEF mRNA level by densitometric analysis. **p < 0.01 vs. control.