FIG. 6.
RelB/p52 NF-κB complexes induce cyclin D1 promoter activity and interact poorly with IκB-α. (A) NMuMG untransformed murine mammary gland cells were transiently transfected, in triplicate, with 1 μg of −1745 bp WT cyclin D1 luciferase gene reporter construct and 0.5 μg of pSV40-β-Gal in the presence of 0.5 μg each of RelB and p52 or p65 and p50 plasmid expression vectors or 1.0 μg of the empty vector. After 48 h, cultures were harvested, normalized for β-Gal activity, and assayed for luciferase activity. Values for luciferase activity, relative to stimulation with empty vector DNA (EV), are presented after normalization to β-Gal activity (norm. luc. activ.). The error bars represent the standard deviations. (B) Nuclear extracts were prepared from MMTV-SR-IκB-α line15 or WT mammary glands at day 14.5 of first pregnancy, and samples (5 μg) were subjected to EMSA for NF-κB binding in the presence of 0, 0.25, or 0.5 μg IκB-α-GST protein, as indicated. Alternatively, samples were incubated overnight at 4°C using an antibody against either p65 or RelB proteins prior to the binding reaction and addition of IκB-α-GST. The positions of bands 1, 2, and 3 are indicated.