Figure 4

Cytokine-induced NF-κB activation is minimally augmented during subsequent TLR2 stimulation. A) IκBα and β-actin cellular protein levels were assessed using immunoblot analysis of extracts from human airway epithelia that were treated without or with media containing TNF-α (100 ng/ml) and IFN-γ (100 ng/ml) for 30 min. B) IκBα and β-actin cellular protein levels were assessed using immunoblot analysis of extracts from human airway epithelia that were first treated without or with media containing TNF-α (100 ng/ml) and IFN-γ (100 ng/ml) for 24 hours, followed by incubation without or with Pam3CSK4 (25 mg/ml) for 30 min. C) IκBα and β-actin protein levels in the experiment outlined in B were quantified using band densitometry of immunoblot analyses results with inclusion of samples from three individuals. Values were calculated as IκBα/β-actin, were normalized to the untreated control value for each individual, and are expressed as mean fold change in IκBα ± S.D. (n = 3 in each group). D) NF-κB-dependent gene activation was assessed using luciferase activity assays of extracts from human airway epithelia that were initially infected with an adenoviral vector expressing a luciferase gene driven by four tandem NF-κB sites. Cells were then treated without or with media containing TNF-α (100 ng/ml) and IFN-γ (100 ng/ml) for 24 hours, followed by incubation with Pam3CSK4 (25 mg/ml) for 24 hours. Values are expressed as mean ± S.D. (n = 2-3 samples from 4 individuals in each group), and a significant difference from the untreated control is indicated by an asterisk.