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Astrocytes represent very suitable imaging substrate for detecting translocating proteins as previously described. Using immunocytochemistry and image analysis, we showed that the treatment of rat primary cortical astrocytes with TNF LDN-57444 ic50 for 15 min and 24 hr resulted in the translocation of the p65 subunit of NFB to the cell nucleus, although to different extent. TNF activation of NFB peaks within 30 min and then fades over time to lower level after hours of continued exposure. Vehicle treated cells after both 15 min and 24 hr showed no indication of p65 translocation. As expected, based on the assay design, none of the compounds caused significant translocation of p65 after 15 min, indicating that most likely they are not activating pathways associated with IB phosphorylation and rapid activation. Seven of the eighteen selected hit compounds Mitochondrion caused p65 nuclear translocation after prolonged exposure, suggesting that our compounds activate NFB in noncanonical fashion. To test whether the selected compounds result in NFB p65 activation and up regulation independently of cytokine receptor activation in neurons, we determined p65 protein levels in cytoplasmic and nuclear fractions following 24 hr treatment with compounds SRI 22772, 22782, and 22820 at the maximum effective concentrations as determined by the analysis shown in Figure 4. As positive control, we treated neurons with 100 ngml of TNF for 30 min. Western blot analysis revealed that IB was markedly decreased in TNF treated neurons, as expected, because of the effect of TNF receptor activation and subsequent phosphorylation of IB. However, compound treated neurons show no changes in IB expression compared with untreated cells or TNF treated cells. p65 protein expression in the cytoplasm was significantly increased following 24 hr of exposure to the compounds. As expected, TNF decreased p65 protein levels in XL888 clinical trial the cytoplasm of primary neurons. Nuclear presence of NFB was significantly increased after treatment for 24 hr with the compounds. As expected, TNF increased nuclear presence of p65. Finally, when the data from the cytoplasm and the nucleus were summed, all compounds caused marked increase of total cellular p65, supporting the view that our compounds achieve p65 activation by overall increased p65 protein synthesis. Primary cultures of neurons were treated at 6 days in vitro for 24 hr with the maximally effective concentration of the compound as determined from our profiling experiments. After incubation with the compounds, immunocytochemistry was performed and cultures were immunostained for p65 and glial fibrillary acidic protein for identification of cell type and DAPI. Indeed, each of the compounds tested, SRI 22772, 22782, and 22820, resulted in significant p65 translocation to the nucleus. Quantification of translocation followed by statistical analysis indicated statistically significant relocation of p65 in the nucleus of primary neurons following compound and TNF treatment.