Figure 6

NFκB is constitutively active in multiple lymphoid cells (A) YT (lanes b-d), quiescent Kit225 (lanes e-g), naïve (lanes h-j) and quiescent activated human PBMCs (lanes k-m) were stimulated with IL-2 for 30 min before nuclear extracts were prepared. Equivalent amounts (5 μg) were used in EMSA reactions employing a STAT5 (upper panel) or NFκB radio-labeled probe (lower panel). A reaction without nuclear extract (free probe, lane a) or with unlabeled probes (lanes d, g, j, m) were used to confirm the specificity of the bands. (B) Constitutive NFκB is composed of p50 and p65 subunits in lymphoid cells. Antibodies to p50 (lane c) and p65 (lane d) subunits of NFκB were used either alone or in combination (lane e) for supershift assays in Kit225 nuclear extracts. Control IgG (normal goat serum, lane f) and a reaction without nuclear extract (free probe, lane a) were used to confirm the specificity of the bands. Star (*) indicates a non-specific band.