Figure 3

RelB protein expression is decreased by cigarette smoke exposure in Normal lung fibroblasts. Lung fibroblasts from Normal, At Risk and COPD subjects were exposed to 2% CSE for 3, 6, or 24 hours and RelB mRNA and protein levels evaluated. (A) RelB mRNA-CSE: RelB mRNA was increased in Normal and At Risk fibroblasts exposed to 2% CSE for 24 hours (*p < 0.05 and **p < 0.001). RelB mRNA levels were not significantly altered by CSE in COPD lung fibroblasts. Results are expressed as the mean ± SEM (fold-change) utilizing fibroblasts derived from 4–6 individual subjects for each group. (B) RelB protein: There was a noticeable decrease in RelB protein in Normal fibroblasts exposed to 2% CSE for 6 hours. Basal RelB protein levels were noticeably less in At Risk and COPD lung fibroblasts compared to Normal fibroblasts. Representative western blots are shown of three experiments. (C) RelB protein- Densitometry: Densitometric analysis of RelB expression revealed a significant difference in basal RelB protein expression between Normal and At Risk and COPD lung fibroblasts in the absence of 2% CSE (media only). RelB protein expression decreased in Normal lung fibroblasts at 6 hours of exposure to 2% CSE compared to media control (* p < 0.05). Results are expressed as the mean ± SEM (fold-change) utilizing fibroblasts derived from three individual subjects for each group. (D) RelB protein: MG-132: Normal lung fibroblasts were pretreated with MG-132 for 2 hours prior to cotreatment with 2% CSE for 6 hours and protein harvested for RelB expression. RelB levels were decreased following exposure to 2% CSE for 6 hours. MG-132 increased basal levels of RelB and prevented the loss of RelB after exposure to 2% CSE. Western blot image is representative of 3 separate experiments.