CSC-induced phosphorylation of ERK1/2 and IL-1β expression. (A) CSC induces ERK1/2 phosphorylation. NHBEs were incubated with the indicated concentrations of CSC for 30 min at 37°C. Whole-cell protein extracts were prepared from cell lysates, aliquots of 10 μg of protein were separated by SDS-PAGE, transferred to nitrocellulose and probed with an antibody to phosphorylated ERK1/2. Blots were then stripped and reprobed for ERK1/2. The band intensities were determined by densitometry and the fold-change relative to control was plotted as means ± SEM. (B) Nuclear localization of phosphorylated ERK1/2 in NHBEs. Cells were incubated with 0.4 μg/ml CSC for 30 min at 37°C, then fixed with paraformaldehyde and probed with antibody to phosphorylated ERK1/2. Visualization was achieved by incubating the cells with HRP-conjugated secondary antibody and bisbenzamidine substrate. Cells were treated with vehicle alone (Control), with CSC (CSC), or with CSC and the MEK inhibitor, PD98059 (20 μM) (CSC + PD98059). (C) Inhibition of ERK1/2 or IκB phosphorylation attenuates IL-1β expression in A549. Cells were treated with 50 μg/ml PD98059 or 1 mM pyrrolidine dithiocarbamate (PDTC) for 30 min then exposed to 0.4 μg/ml CSC for 60 min. RT-PCR was performed on RNA extracted from these cells using IL-1β-specific primers. Band densities were normalized to GAPDH levels, expressed as fold change relative to untreated controls, and plotted as mean ± SEM for n = 3. The * indicates significance at P < 0.05. CSC = cigarette smoke condensate; ERK = extracellular signal-regulated kinase; IκB = inhibitor of NFκB; MEK = MAPK kinase; NBHE = normal human bronchial epithelial cell.