Different octamer-specific DNA binding complexes in human dendritic cells and macrophages.

(A) EMSAs with nuclear proteins from monocytes (day 0, lanes 1 and 4), immature dendritic cells (day 7, lane 2), and mature dendritic cells (day 10, lane 3). As a control for specificity of DNA binding, an excess of an unlabeled octamer binding probe was added to nuclear proteins from monocytes (C1, lane 4). The 3 detectable octamer-specific complexes are designated I to III (see below). The asterisk indicates a prominent unspecific band, and the circle marks another unshiftable complex, probably containing Oct-2. As shown by supershift EMSAs (see B), the 3 detectable octamer-specific DNA complexes are composed of Oct-1 (complex I) and different isoforms of Oct-2 (complexes II and III). The signals of the free probe are cut off. (B) Supershift EMSAs with nuclear proteins from monocytes (day 0, lanes 1-3), immature dendritic cells (day 7, lanes 4-6), and mature dendritic cells (day 10, lanes 7-10). For supershift assays, 1 μl of each of the octamer-factor–specific antibodies was added as indicated (lanes 2 + 3, 5 + 6, and 8 + 9). As a control for specificity of the supershifts, 1 μl of NRS was added to nuclear protein from mature dendritic cells (lane 10). (C) EMSAs with nuclear proteins from monocytes (day 0, lane 1), macrophages (lane 2), and macrophages treated with monocyte-conditioned medium for another 3 days (Mph+, lane 3). The specificity of DNA binding was controlled by addition of an excess of an octamer-specific probe (C1, lane 4) and of a κB-specific probe (C2, lane 5) to nuclear extracts from Mph+ cells. (D) Supershift EMSAs with nuclear proteins from macrophages (lanes 1-3) and macrophages treated with monocyte-conditioned medium for another 3 days (Mph+, lanes 4-7).