To determine whether STAT3 could bind the Bortezomib Proteasome inhibitor Necdin promoter in intact cells, chromatin immunoprecipitation assays were performed in 3T3 v-Src cells using an antibody specific to STAT3. As shown in Figure 2D, PCR yielded Necdin promoter DNA immunoprecipitated with an anti-STAT3 antibody in the region of the 2558 putative STAT3-binding site, but not at a control location on the NDN promoter. The specificity of this binding interaction is demonstrated by the lack of signal generated when a control antibody is used . These data provide evidence that STAT3 can directly bind the Necdin promoter in intact 3T3 v-Src cells. Since both the EMSA and ChIP assays suggest that STAT3 has the ability to bind to the NDN promoter both in vitro and in vivo, this provides further evidence that control of NDN expression by STAT3 occurs through a direct binding event at the promoter and that gene regulation primarily occurs at the level of transcription. We next examined whether down-regulation of Necdin occurred in human tumor cells with activated STAT3. Expression of Necdin has been previously shown to be repressed in melanoma cells so we examined whether this had a correlation with STAT3 activity. STAT3 phosphorylation, STAT3 DNA-binding activity and total STAT3 levels have been shown to increase in A375 melanoma cells in a density-dependent manner in the absence of ligand . A375 cells were plated at increasing density and allowed to grow for 72 h. Nuclear extracts were prepared and analyzed by EMSA. Figure 3A shows that DNA-binding of STAT3 increases with cell density as expected. We then analyzed total protein by Western blot for Necdin expression. Figure 3B shows that expression of total STAT3 and STAT3 phosphorylation is up-regulated in a density-dependent manner. Conversely, as STAT3 activation increases, Necdin expression is down-regulated at the protein level. To confirm that the repression of Necdin expression is STAT3- dependent, A375 cells were plated at high density, and allowed to adhere overnight before being treated with either DMSO or the STAT3-inhibitor CPA-7 for 24 h . Western blot analysis shows that when A375 cells are plated at low density , Necdin expression is high, whereas activated STAT3 levels are low . Cells plated at high density , show higher levels of p-STAT3 and decreased expression of Necdin. Treatment of high density A375 cells with CPA-7 for 24 h inhibited STAT3 activation , and Necdin levels in these cells are restored to high levels, comparable to cells plated at low density. IL-6 acts as an autocrine growth factor in prostate cancer and has been linked to progression of tumors . IL-6 signals are transmitted via the JAK-STAT pathway from receptors on the cell surface to the target genes in the nucleus, involving phosphorylation and activation of STAT3 .