Influence of NOD on TNF-α mediated NFκB activation.

<p>(A) HUVECs were stimulated for different time periods with 10 ng/ml of TNF-α. During stimulation NOD (50 µM) was absent (−) or present (+). Nuclear extracts were prepared and assessed for NFκB activation by means of EMSA. (B) In separate experiments the influence of NOD on the degradation of IκBα was assessed. HUVECs were stimulated for different time periods in the presence (+) or absence (−) of 50 µM of NOD. HUVECs grown in medium served as control. IκBα degradation was assessed by western blotting. (C) HUVECs were stimulated for 24 hrs with 10 ng/ml of TNF-α (TNF-α) in the presence (+) or absence (−) of 50 µM of NOD. Cells that were left untreated (medium) served as control. Nuclear- and cytoplasmic extracts were prepared and assessed for the expression of phosphorylated p65 at Ser276 (p65- Ser276) and total p65 (p65) by western blotting. GAPDH was used as loading control. Note that in the condition of TNF-α+NOD phosphorylation of p65 at Ser276 is not detectable in the nuclear extracts and that expression of total p65 in both nuclear and cytoplasmic extracts is decreased. The results of a representative experiment are shown. A total of 4 independent experiments with different HUVEC cultures were performed.