FIGURE 4.

LIGHT and LTα1β2 induce nuclear translocation of classical and noncanonical NF-κB proteins in EC. A, Nuclear extracts prepared from HUVEC incubated with either TNF (10 ng/ml), LIGHT (100 ng/ml), or LTα1β2 (100 ng/ml) for the times indicated were immunoblotted using anti-p65, anti-p100/p52, anti-RelB, or anti-histone-3 (loading control) as indicated. B, Nuclear extracts of HUVEC treated with either TNF (10 ng/ml; left) or LTα1β2 (100 ng/ml; right) were subjected to EMSA using a γ-³²P[ATP]-labeled NF-κB consensus oligonucleotide probe. Samples in lanes 4 – 6 and lanes 10–12 were incubated with anti-p65 (2 μg/sample) in the EMSA reaction and the supershifted (SS) complex in these lanes is indicated. Samples in lanes 1–3 and lanes 7–9 were incubated with an isotype-matched nonspecific Ig (2 μg/sample). C, Supershift analysis was performed as described in B using anti-p52. The panel at the far right (lanes 13–18) is a longer exposure of the LTα1β2 experiment in the middle panel (lanes 7–12).