Levels of these proteins started to increase after 8 hours peaking at 12 16 hours after NGF withdrawal. Trib3 was localised in both the nucleus and cytoplasm, whereas Ddit3 was localised mainly in the nucleus after NGF with drawal. However, in the presence of CEP 11004, the levels of both proteins were decreased significantly to almost basal levels and more importantly were not detected in the nucleus. The protein levels of the other three genes, Txnip, Ndrg1 and Mxi1 were also studied by immunoblot ting and immunofluorescence. Significant but modest increases in the levels of the Txnip, Ndrg1 and Mxi1 proteins were seen after NGF withdrawal and CEP 11004 reduced this to varying degrees. The increase in Txnip protein level after NGF withdrawal was smaller than that seen at the transcriptional level.

The effect of CEP 11004 was also not as significant at the protein Inhibitors,Modulators,Libraries level. The increase in the level of the Txnip protein and its loca lisation after NGF withdrawal were also studied by immu nofluorescence. The Txnip protein was clearly seen at Inhibitors,Modulators,Libraries 8 hours after NGF withdrawal in both the nucleus and cyto plasm and this was followed by a steady increase in pro tein levels over time. Both of the Myc pathway associated proteins, Ndrg1 and Mxi1, also increased in level after NGF withdrawal and CEP 11004 reduced this increase. The txnip and trib3 promoters contain potential c Jun binding sites We previously showed that three of the genes that are induced after NGF withdrawal in sympathetic neurons, c jun, dp5 and mkp1, are direct targets of c Jun.

The induction of these genes after NGF deprivation is strongly reduced by CEP 11004 and the c jun, dp5 and mkp1 promoters contain functionally important ATF sites that have been shown to bind c Jun ATF2 heterodimers in chromatin immunoprecipitation assays and EMSA experiments. Some of the induced genes identified Entinostat in our exon array analysis might also be direct targets of c Jun, in particular those whose mRNA induction after NGF withdrawal is strongly sup pressed by CEP 11004, for example txnip and trib3. We therefore searched for Inhibitors,Modulators,Libraries conserved potential c Jun binding sites in the promoter, first exon and first intron of the rat txnip and trib3 genes. The txnip promoter contains an ATF site, 919 bp upstream of Exon 1 in the rat gene, that is identical in sequence to the reverse comple ment of the jun2 TRE site in the c jun promoter.

This site is conserved in the rat, human, and cow txnip genes and contains two base changes in the mouse gene. In the case of trib3, we identified a conserved ATF site 14 bp upstream Inhibitors,Modulators,Libraries of Exon 1 in the rat gene. This site is identical to the reverse complement of the ATF site in the dp5 promoter and is conserved in the rat, mouse and cow genes and only one nucleotide differs in the human trib3 gene.