The genes identified may serve as primary effectors of Nkx2-1 functions in different developmental cell contexts. We determined expression levels of Nkx2-1 target genes identified in development and correlated their expression to the level of NKX2-1 in more than ten public human lung tumor data sets. The regulatory networks discovered clarify the diverse biological roles of Nkx2-1 observed in development, and provide a rationale for the association of NKX2-1 levels and NSCLCs prognosis via its downstream targets. The monoclonal antibody detects nuclear Nkx2-1 protein expression while the polyclonal antibody detects signal both in the nucleus and cytoplasm. Detection of Nkx2-1 in the cytoplasm has been previously shown by other authors. These results indicate that lung epithelial cells expressing Nkx2-1 in early and late lung development go through different biological processes; it is likely that Nkx2-1 elicits different functions by binding to specific target genes in these developmental stages. Context specific gene regulation controlled by Nkx2-1 has been shown in early and late brain development, where Nkx2-1 regulates the specification of interneuron subtype in early proliferating telencephalic progenitors, and later, the migration and sorting of post mitotic neurons to different TWS119 GSK-3 inhibitor regions of the brain. We also characterized, by western blots, Nkx2-1 protein expression patterns at different mouse lung developmental stages, thyroid, liver, and mouse MLE15 and human H441 cell lines. Using the rabbit polyclonal Nkx2-1 antibody, the same antibody used in ChIP assays, we detected two major bands between 40 and 50 kD in E13.5, E19.5, and adult lung, and in the MLE15 lung epithelial cell line. The higher molecular weight or lower mobility band was the major band detected at E11.5. Its abundance was increased from E13.5 to E19.5 consistent with previous reports. Only one band corresponding to the lower mobility protein was detected in mouse WZ4002 thyroid and in the H441 human lung carcinoma cell line although their mobility is not identical. Two mouse Nkx2-1 transcripts differentially expressed during lung development are translated in vitro into two proteins that share a common DNA binding domain but differ in their Nterminal domain. The function and regulation of these isoforms in lung development are unknown, although they differentially activate the surfactant protein C promoter. Also Nkx2-1 posttranslational modifications such as phosphorylation, acetylation and redox state can modify Nkx2-1 proteins altering their molecular weight, mobility in electrophoresis assays and transcriptional activity ; the lack of specific antibodies for each isoform or posttranslational modification precludes evaluating them in the present studies.