n -6 demonstrated to be critical for p65-driven gene expression of many target genes. The IKK complex is known to phosphorylate p65 at serine 536, in this way regulating p65 nuclear localization. We assessed whether N. vitripennis venom affects these early LPS-induced events in R-7128 site Raw264.7 cells. We found that LPS promoted phosphorylation of IKK in its activation loop that was apparent after 5 minutes and lasted up to 15 minutes of LPS treatment. Venom pretreatment did not affect IKK phosphorylation. Next, it was investigated whether the venom alters LPS-induced phosphorylation of p65. As the Western blot analysis in figure 3 shows, p65 phosphorylation in Raw264.7 cells is highly increased after 15 minutes LPS induction. However, no difference in LPS-induced p65 phosphorylation was apparent after venom co-treatment. d. Venom does not interfere with nuclear translocation of the p65 subunit. LPS induction in Raw264.7 macrophage cells is a pleiotropic cytokine involved in acute phase and immune reactions and inflammatory responses, the expression of which in response to inflammatory stimuli, strongly depends on NF-kB. Macrophages are key players in the inflammatory response, and they secrete high levels of IL-6 upon ligation of Toll-like receptors by pathogen-associated molecular patterns. We therefore explored the effect of N. vitripennis venom on TLR4-dependent, LPS-induced IL-6 production in Raw264.7 mouse macrophages. As shown in figure 2A, the elevated levels of IL-6 protein detected after 6 hours LPS treatment were strongly repressed in the presence of the venom in a concentrationdependent manner. Nasonia vitripennis venom pretreatment also inhibited LPS-induced IL-6 mRNA levels, yet this effect was less prominent and required ten-fold higher doses of venom. c. Venom does not interfere with canonical NF-kB signaling in the cytoplasm. One of the major mechanisms leads to canonical NF-kB activation, that is followed by nuclear translocation of the NF-kB p65 subunit prior to NF-kB/DNA binding. Therefore, the level of p65 was measured in nuclear extracts of Raw264.7 cells either untreated or venom-treated for 10, 30 or 60 minutes LPS induction. Antibody against Poly ADPRibose PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19657107 polymerase was used as a loading control for the nuclear fractions. When analyzed by Western blotting, LPSinduced p65 nuclear translocation was not affected by N. vitripennis venom. These results were confirmed in figure 4B by immunofluorescence staining to visualize the trafficking of the p65 subunit. LPS induction after 30 and 60 minutes caused an accumulation of green fluorescent signal in the nucleus, conform the translocation of p65 from the cytoplasm to the nucleus. Coincubation 
of these cells with venom, however, did not alter this translocation. e. Venom does not interfere with NF-kB transactivation independent from the promoter context. Since no influence involved in the transcriptional activation of NF-kB is the activation of the IKK kinase complex and the concomitant phosphorylation and degradation of the IkBa protein, which allows the release of free NF-kB and its subsequent translocation to the nucleus. Furthermore, post-translational modifications of p65 have been of N. vitripennis venom was apparent on the subcellular localization of p65 NF-kB following inflammatory stimuli, the next step was to investigate whether the observed inhibition of NF-kB activity could be explained by a nuclear phenomenon. To approach this question, it was investigated whether