Figure 5. HO-1 induction by NOD is not required for inhibition of NFκB.

(A) HUVECs were pre-treated for 2 hrs with 5 µg/ml of cyclohexamide (+ CyHx) or left untreated (− CyHx). Hereafter, cells were stimulated for 8 hrs with 10 ng/ml of TNF-α in the presence (+) or absence (−) of 50 µM of NOD. In case the cells were pre-treated with CyHx, this was present during the whole period of stimulation. In case cells were not treated with CyHx, this was absent during stimulation. Western blotting of the cytoplasmic fractions revealed that de novo protein synthesis was effectively inhibited by CyHx. Note that VCAM-1 is not induced by TNF-α in the presence of CyHx. Also in the combination of TNF-α+NOD the induction of HO-1 did not occur. GAPDH was used as loading control. (B) Nuclear extracts were prepared and assessed for NFκB activation by means of EMSA. Specificity of the bands was assessed by adding an excess of unlabelled NFκB consensus (cold consensus (CC)) or mutated (cold mutated (CM)) oligonucleotides to the samples. To demonstrate the presence of p50 and p65 in the shifted bands super-shifts (SS) were performed by adding anti-p50 or anti-p65 monoclonal antibodies to the samples. In A und B the results of a representative experiment are shown. A total of 4 independent experiments with different HUVEC cultures were performed.