Fig. 8.
NF-κB subunits are recruited to κB genomic response elements within the CXCL1 and CXCL2 gene promoters in a signal-dependent and IκBα-sensitive manner. A: 832/13 cells were stimulated with 1 ng/ml IL-1β for 5, 10, and 15 min or 100 U/ml IFNγ for 5 and 10 min. Whole cell lysates (40 μg) were immunoblotted for PO4-IκBα, total IκBα, PO4-STAT1, and β-actin (as loading control). B: 832/13 cells were treated for 15, 30, and 60 min with 1 ng/ml IL-1β. Whole cell lysates (20 μg) were blotted for abundance of total IκBα with β-actin serving as loading control. C–F: 832/13 cells were cultured with recombinant adenoviruses that express either βGAL or IκBαSR for 12 h. Cells were then stimulated for 15 min with 1 ng/ml IL-1β. ChIP assays were performed with antibodies that immunoprecipitated p65 (C, E) and p50 (D, F). Proximal (C–F) NF-κB elements in the CXCL1 and CXCL2 core promoters were targeted for real-time PCR amplification. **P < 0.01 vs. βGAL + IL-1β. Data in C–F represent means ± SE from 3 individual experiments; immunoblots shown in A and B are representative of 2 independent experiments.