Sickle mononuclear leukocytes activate NF-κB in MVEC.

Mononuclear leukocytes (15 leukocytes/endothelial cell) or TNF-α (10 ng/mL) were incubated with MVEC for 1 hour. After incubation, MVEC nuclear proteins were extracted for NF-κB analysis. Nuclear extracts (10 μg protein) were incubated with 2 ng [³²P]-labeled dsDNA containing a consensus NF-κB sequence and analyzed by electrophoretic mobility shift assay. Lane 1, [³²P]-labeled dsDNA probe alone; lane 2, control (media only)-treated MVEC; lane 3, TNF-α–treated MVEC; lane 4, normal mononuclear leukocyte-treated MVEC; lane 5, sickle mononuclear leukocyte-treated MVEC. Results are from a single representative experiment of 2.