To fully understand the differences in binding patterns in different cell contexts we will need to identify the consensus sequences and the chromatin modifications of the binding sites in genomic regions only bound at E11.5 or only bound at E19.5. Future analyses will be focused in discerning these alternatives and characterizing Nkx2-1 binding sites in different contexts. Finally, we observed strong binding to brain and thyroid genes not expressed at detectable BU 4061T Proteasome inhibitor levels in developing lung, suggesting that binding of Nkx2-1 does not imply activation of transcription. In certain cases, binding may precede activation such as in the case of Sftpa and may prime the gene for activation upon recruitment of other transcription complexes and/or co-factors to the promoter. The identification of unique Nkx2-1 targets at E11.5 and E19.5 will facilitate the evaluation of possible mechanisms that control specificity. Overall, we provide novel insights into biological processes regulated by Nkx2-1 in different cell contexts in development, and cancer. We identified Nkx2-1 direct target genes in mouse lung epithelium that are primary effectors of Nkx2-1 functions, in particular cell proliferation genes. We showed that expression levels of the target genes depend on NKX2-1 levels in NSCLC. NKX2-1 has been associated to longer, similar or shorter patient survival in NSCLC, depending on expression levels. Therefore, evaluation of NKX2-1 expression levels relative to its downstream targets will provide a way to sub-classify NSCLCs, and understand the mechanisms underlying associations to patient survival. The later did not work in ChIP assays in the conditions tested. Although some antibodies work well in experiments such as western blots, or immunocytochemistry, they may not necessarily work in ChIP assays since the fixative conditions used may mask or destroy some epitopes. Monoclonal antibodies, such as ab76013, have higher specificity than polyclonal sera, but polyclonal sera, such as 07-601, may recognize several epitopes of the target, increasing signal levels. We selected the rabbit polyclonal Nkx2-1 antibody for the ChIPchip analyses based on our BI-D1870 previous results and additional experiments performed for this manuscript. We have previously shown specificity of this antibody in ChIP-PCR analyses in vitro and in vivo. Briefly MLE15 cells were transfected with a wild type or mutant Sftpb promoter construct containing mutations of four Nkx2-1 consensus sites. ChIP-PCR assays were performed with the rabbit polyclonal Nkx2-1 antibody or IgG. Mutation of the Nkx2-1 binding sites abolished binding of Nkx2-1 to the promoter and therefore no PCR band is observed when the mutant DNA is immunoprecipitated with the Nkx2-1 antibody. PCR with oligonucleotides in the b-actin locus were also used to indicate the absence of non-specific binding.