Electrophoretic mobility shift assay for NF-κB.

Lane 1, positive control with TNF-treated microvascular endothelial cells. Lanes 3 and 10 show a weak NF-κB band in mice pretreated withEscherichia coli LPS and D-galactosamine, a combination that stimulates murine NF-κB activation. The sickle mouse kidney reveals a strong NF-κB band after H/R (lane 4), which is not present in the sickle mouse at ambient air (lane 2). The band on lane 4 is identified as NF-κB by the ability to competitively inhibit it with unlabeled NF-κB consensus oligonucleotide (lane 5). Sickle mouse liver at ambient air shows a faint NF-κB band (lane 9) that is much more prominent in the mouse liver after H/R (lane 11). This band is competitively inhibited by unlabeled NF-κB consensus oligonucleotide (data not shown). Antibody to the cRel subunit of NF-κB does not cause a supershift in sickle mouse liver or kidney. There is no NF-κB band demonstrable in the normal mouse liver or kidney with or without H/R. Hb SS indicates sickle mouse; LPS, Escherichia colilipopolysaccharide plus D-galactosamine; cold wt comp, unlabeled NF-κB consensus oligonucleotide probe; anti-cRel, antibody to cRel subunit of NF-κB; MVEC, microvascular endothelial cell; TNF, tumor necrosis factor; amb air, ambient air; H/R, hypoxia and reoxygenation.