Increased nuclear translocation of Rel proteins during in vitro generation of human dendritic cells and macrophages results in an increase in κB-specific DNA binding activity.

(A) EMSAs performed 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 κB-specific probe (C, lane 4) was added to nuclear proteins from monocytes. The 3 detectable κB-specific DNA binding complexes are designated I through III (see below). The asterisk indicates a complex of unknown composition. The signals of free probes were cut off. (B) Supershift EMSAs were performed with nuclear proteins from monocytes (day 0, lanes 1-6), immature dendritic cells (day 7, lanes 7-12), and mature dendritic cells (day 10, lanes 13-19). For supershift EMSAs, 1 μl of each of the Rel protein–specific antibodies was added to the extracts as indicated (lanes 2-6, 8-12, and 14-18). As a control for the specificity of supershifts, 1 μl of normal rabbit serum (NRS) was added to nuclear extracts from mature dendritic cell (lane 19). The 3 indicated κB-specific DNA complexes are composed of p50 homodimers (III) and RelB/p50 and RelB/c-Rel heterodimers (II). Complex I consists of 2 NF-κB complexes: p65/p50 and p65/c-Rel heterodimers. The asterisk indicates a complex of unknown composition. (C) EMSAs using nuclear proteins from monocytes (day 0, lane 1), differentiated 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 unlabeled κB-specific probe to nuclear extracts from monocytes (C, lane 4). (D) Supershift EMSAs using nuclear proteins from macrophages (lanes 1-6) and macrophages treated with monocyte-conditioned medium for another 3 days (Mph+, lanes 7-13).