In addition we showed using the same antibody that Nkx2-1 binds to the endogenous Sftpb promoter in the lung but not the thyroid where the DNA in this region is highly methylated. This same antibody shows binding of Nkx2-1 to the thyroglobulin gene promoter in both tissues but not to b-actin. Supporting experiments that show the specificity of the antibody are included in the results MK-0683 section where down VE-821 regulation of Nkx2-1 gene expression by shRNAs results in reduction in intensity of the two major bands in Western blots. Those shRNAs do not reduce the levels of the non-specific protein b-actin. Some members of the cytohesin and EFA6 families of Arf-GEFs are stabilised on their target membranes by the contribution of their PH domains, which interact with specific phosphoinositides. To determine whether Yel1 can also localise to the membrane in this manner, a strain was constructed in which residues 1�C387 were replaced with GFP in the genome of an arf3D mutant. This truncation removes the Sec7 domain leaving only the C-terminal portion of the protein that contains the PH domain. At steady state GFP-Yel1 was undetectable at the plasma membrane and was found instead throughout the cytoplasm. These data suggest that, although Yel1 contains a recognisable PH domain, its affinity for phosphoinositides is insufficient to allow the protein to stably associate with the plasma membrane. Interestingly a study by Mark Lemmons�� group in which the activities of all PH domains from yeast were assessed using a Ras activation assay demonstrated that, under certain conditions, a Ras-Yel1-PH domain fusion was capable of targeting Ras to the membrane, where it could rescue the defect of a cdc25ts mutant strain. However when this construct was tagged with GFP and localised in the cell it was not found at the membrane but instead targeted to the nucleus. These observations suggest that the PH domain of Yel1 is able to associate weakly with membranes but that the degree of membrane association is undetectable at the level of the light microscope. The results also imply that in the fulllength protein, the N-terminus, perhaps through the activity of the Sec7 domain, is additionally required to ensure that a stable interaction with the membrane is sustained. We next examined the localisation of Arf3 in the GFP-Yel1 mutant yeast. As expected, Arf3-RFP was displaced from the plasma membrane in this strain. Since the isolated C-terminal portion of Yel1 is not capable of stably localising the protein to the membrane, we next asked whether the Sec7 domain can perform this function. To address this, a yel1D mutant strain in which endogenous Arf3 is tagged with GFP was transformed with plasmid-borne copies of RFP-Yel1 or RFP-Yel1, the latter plasmid encoding only the Sec7 domain.