Phosphorylation of IκB and expression of NFκB-luciferase reporter gene in cells exposed to CSC. (A) CSC exposure results in phosphorylation of IκB in NHBEs. Cells were grown to approximately 80% confluency then treated with the indicated concentrations of CSC for 30 min at 37°C. Whole-cell protein extracts were prepared and 10 μg aliquots were separated by SDS-PAGE, blotted and probed with an antibody to phosphorylated IκB. The blot was then stripped and reprobed for IκB. The band intensities for phosphorylated IκB were determined by densitometric scanning and normalized to the levels of IκB in each lane. Values are means ± SEM for n = 3. (B) CSC-induced immunofluorescent staining for phosphorylated IκB in NHBEs. Cells were treated with vehicle (Control) or 0.04 μg/ml CSC (CSC) for 30 min at 37°C. Cells were fixed with paraformaldehyde and probed with antibody to phosphorylated IκB. (C) and (D) Effect of CSC on expression of an NFκB-regulated luciferase reporter gene in A549 cells. Cells were transfected with a plasmid containing an NFκB binding site upstream of a luciferase reporter sequence. The transfected cells were exposed to 0.4 μg/ml CSC for 30 min at 37°C (C) or increasing doses of CSC (D) and lysates were assayed for luciferase activity using a luminometer. Differences in transfection efficiency were normalized by cotransfection with a LacZ-containing plasmid. The treatments were performed in triplicate and expressed as mean ± SEM for n = 3. CSC = cigarette smoke condensate; IκB = inhibitor of NFκB; SEM = standard error of the mean; NFκB = nuclear factor-kappa B; NHBE = normal human bronchial epithelial cell.