Constitutive NF-κB activity correlates with Blk expression. (A) WB analysis of p65, p50, and IκB-α in cytoplasmic (c) and nuclear (n) extracts of Jurkat T cells either not stimulated (−) or stimulated for 1 hour with 100 ng/mL TNF-α and unstimulated (−) malignant CTCL cells (MF2000). (B) MF2000 cells were cotransfected with a luciferase reporter construct containing either no insert (pGL3), a NF-κB consensus binding site (pGL3-NF-κB), or the proximal VEGF promoter (pGL3-VEGF) and a Renilla luciferase vector. At 48 hours after transfection the cells were lysed, and the luciferase activities were determined. The coexpressed Renilla luciferase activity was used for normalization of transfection efficiency. The experiment was performed with triplicate cultures and is representative of 3 independent experiments. (C) Jurkat, MF2000 (malignant), and MF1850 (nonmalignant) cells were incubated for 3 hours with TNF-α (100 ng/mL), Mg-132 (10 μM), LckI (10 μM), Jak3I (40 μg/mL), or vehicle (−) as shown. Then, DNA binding p65 and p50 were affinity purified from nuclear lysates using bio-oligos with a sequence identical to a putitative NF-κB binding site in the Blk promoter. Finally, the levels of precipitated p65 and p50 were analyzed by WB. (D,E) MF2000 cells were incubated for 24 hours in media (Med.) containing vehicle (−), Jak3I (40 μg/mL), NF-κBI (100 nM), LckI (10 μM), JNKI (5 μM), Ag1478 (200 ng/mL), or increasing concentrations (→) of the NF-κB inhibitors JSH-23 (10, 20, 40 μM) and Mg-132 (0.4, 4, 40 μM). Subsequently, the expression of Blk and Erk was determined by WB.