N. vitripennis venom could interfere with NF-kB transactivation, independent from the promoter context. Therefore, its effect on the transcriptional activation of a Gal4-p65-dependent reporter gene, transactivated by the p65 chimera, was analyzed. This Gal4-p65 fusion protein, composed of the DNA-binding domain of the yeast nuclear protein Gal4 and the transactivation domain of the NF-kB subunit p65, stimulates p2-50hu.IL6P-luc+, a luciferase reporter gene, preceded by two Gal4-binding DNA sequence elements and the minimal IL-6 promoter. In this particular nuclear setup, no direct influence of other responsive elementbound transcription factors, which are normally present in the IL6 promoter context, or interference of cytoplasmic events need to be taken into account. When the glucocorticoid receptor, a ligand-activated transcription factor present in the cytoplasm, binds to its agonist, e.g. the synthetic glucocorticoid dexamethasone, the receptor translocates to the nucleus and interferes with the transactivating function of NF-kB, in a process called transrepression. Cells were incubated with DEX as a purchase 518303-20-3 positive control for transrepression of the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19654567 NF-kB pathway. In these experiments, HEK293T cells that are devoid of GR, were transiently transfected with the expression constructs, and treated with venom, 
DEX or were left untreated. Finally, GAL4p65 driven expression of luciferase was quantified based on the measurement of its activity. Whereas Gal4 protein on its own cannot activate the reporter, overexpression of the nuclear fusion protein Gal4-p65 clearly activated reporter gene expression, as Anti-Inflammation by Venom from Nasonia vitripennis expected. After adding DEX to the cells, the level of transactivation by the nuclear fusion protein Gal4-p65 could be specifically repressed, when sufficient exogenous GR was present. However, repression of Gal4-p65-induced transcriptional activation could not be achieved by the N. vitripennis venom, indicating that compounds in the venom do not interfere with the transactivation potential of the p65 subunit in the nucleus or that they act on specific components of the transcriptional machinery that is not involved in this Gal4 assay. 2. Effect of venom on MAPK activation pathways: venom causes prolonged JNK-activation It is well established that the phosphorylation and, in turn, activation of MAPKs, i.e. p38, ERK1/2 and JNK, are crucial for LPS-induced AP-1 and NF-kB activation and subsequent 4 Anti-Inflammation by Venom from Nasonia vitripennis and figure S5). However, after 60 minutes LPS induction, prolonged JNK activation could be observed when N. vitripennis venom was added to the cells. 3. Effect of venom on NF-kB regulated secondary antiinflammatory processes Next to the well-studied direct regulation of NF-kB signaling, the regulatory networks that control the NF-kB response are extensive. The best studied and well accepted mechanism for termination of the NF-kB response involves the resynthesis of IkBa proteins induced by activated NF-kB. Newly synthesized IkBa can enter the nucleus, remove NF-kB from the DNA, and relocalize it to the cytosol. Another important negative regulator of the NF-kB response is A20, which is up-regulated following NF-kB activation. Subsequently, A20 down regulates NF-kB through its dual function as a deubiquitinase and ubiquitin ligase. Furthermore, glucocorticoids are also negative regulators of NF-kB activation and are widely used as antiinflammatory agents