Constitutive NF-κB and SAPK-2/p38 activation by immortalized HUVECs expressing tmTNF.

(A) Constitutive NF-κB activation in immortalized HUVECs expressing tmTNF (HUE_tmTNF) but not in control transfected cells (HUE_Co) as assessed by NF-κB reporter assays (empty bars). The effect of soluble TNF (10 ng/mL) on NF-κB activation is also depicted (shaded bars). Shown are the means and the SD of a minimum of 3 independent experiments (with 5 clones in case of the HUE_tmTNF and 4 clones in case of the HUE_Co). (B) Continuous SAPK-2/p38 activation in tmTNF-expressing HUE cells as determined by Western blot analysis. The effects of pretreatment (6 hours) with either 30 μg/mL TNF-specific mAb T1 (T1) or the SAPK-2/p38–specific inhibitor SB203580 (SB) on HUE_tmTNFcells and of 10 ng/mL soluble TNF (20 minutes) on HUE_Cocells on phosphorylation (pp38, upper panel) and on protein expression (p38, lower panel) of SAPK-2/p38 using the appropriate antibodies (as described in the text) are shown.