HOSCN activates the p65/p50 and p65/c-Rel heterodimeric forms of NF-κB. (A) EMSA analysis of NF-κB activation. HUVECs were stimulated with 10 μg/mL LPS or 150 μM of the designated oxidant as described in the legend to Figure 2 for 1 hour prior to extraction of nuclear proteins. Five-microgram aliquots of nuclear extracts were incubated with radiolabeled NF-κB consensus binding sequence oligonucleotide and separated on 5% nondenaturing polyacrylamide gels. The mobility of the shifted NF-κB consensus probe is shown by the arrow; the slower migration of supershifted bands is shown in brackets. The lanes designated “consensus” show the effect of adding 10-fold excess unlabeled probe; “mutant,” the effect of adding 10-fold excess unlabeled mutant NF-κB consensus oligonucleotide; and “p65 Ab,” “p50 Ab,” and “c-Rel Ab,” the effects of adding polyclonal antibodies to the designated proteins. (B) As in panel A, except HUVECs were incubated with the stimulant for 2 hours, the loading of 10 μg nuclear extract protein/lane, and use of a radiolabeled oligonucleotide probe, derived from the authentic TF-κB–like sequence. (C) Immunofluorescent microscopy localization of p65 in HUVEC monolayers exposed to 10 μg/mL LPS or 50 μM HOSCN in M199 medium with 5% FBS for 4 hours, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100. p65 was visualized using a rabbit anti-p65 polyclonal primary antibody and an FITC-conjugated goat antirabbit IgG secondary antibody by immunofluorescence microscopy (magnification, × 400). (D) Western blot analysis of the nuclear extracts used for 1 hour (left panel) and 2 hours (right panel) using polyclonal antibodies specific for the designated proteins with SP1 as a loading control. For IκB-α only, cytosolic rather than nuclear extracts were analyzed with actin as a loading control.