Research Paper Volume 11, Issue 21 pp 9310—9327

Baicalein attenuates OVA-induced allergic airway inflammation through the inhibition of the NF-κB signaling pathway

Figure 9. Baicalein inhibits TNF-α-induced NF-κB activation in BEAS-2B cells. BEAS-2B cells were pretreated with vehicle control (DMSO) or Baicalein (2.5 μM) for 30 min, followed by exposure to TNF-α (10 ng/mL) for 60 min. Total proteins were extracted and analyzed for P- IκBα (A) and IκBα (B) expression by western blot analysis, with β-actin used as the internal control. After BEAS-2B cells were exposed to TNF-α (10 ng/mL) for 2 h, the nuclear and cytosolic proteins were separated using a cytoplasmic and nuclear protein extraction kit, and the nuclear (C) and cytosolic (D) p65 levels were determined by western blot analysis. β-actin and Lamin B were used as internal controls. (E) p65 staining was carried out, and p65 levels were detected by Cy3-conjugated secondary antibody (red). Cells were counterstained with DAPI (blue) and are shown with a scale bar indicating 50 μm. (F) Relative nuclear immunostaining intensity of p65 was quantified. (mean ± SEM of more than three independent experiments; ##P < 0.01 compared with control, *P < 0.05 compared to TNF-α, **P < 0.01 compared to TNF-α).