PINCH1 protein is predominantly localized at the focal adhesion sites of the periphery of MLN4924 Metabolic Enzyme/Protease inhibitor spreading cells, a pattern overlapping with other focal adhesion proteins, such as ILK and paxillin. Earlier studies show that the affinity for binding of PINCH1 to ILK is reduced in cultured podocytes after injury induced by TGF-b1. However, it remains completely mysterious whether PINCH1 changes its sub-cellular localization after injury; and if so, what are the functional consequences in podocytes. In this study, we demonstrate that PINCH1 is induced and undergoes nuclear translocation in podocytes after TGF-b1 treatment. Furthermore, we have shown that PINCH1 interacts with WT1, which leads to suppression of the WT1-mediated podocalyxin gene expression. Our data identify nuclear transcription factor WT1 as a novel binding partner for PINCH1, and provide novel insights into the mechanism of podocyte dysfunction under pathological conditions. The finding that PINCH1 can shuttle into the nucleus prompted us to investigate its potential function in podocytes. In view of the structural characteristics of PINCH1, which contains five LIM domains that mediate protein-protein interactions, we reasoned that PINCH1 might interact with other nuclear proteins that are important for podocyte biology. Along this line, we found that PINCH1 could interact with WT1, a transcription factor that is exclusively expressed in podocytes in adult kidney. As shown in Figure 5A, when Flag-tagged PINCH1 and GFP-tagged WT1 were co-expressed in podocytes, PINCH1 could be detected in the immunocomplexes precipitated by anti-GFP antibody. In reciprocal experiments, after transfection of podocytes with Flag-tagged PINCH1 expression vector, endogenous WT1 was found in the immunocomplexes precipitated with anti-Flag antibody. These interactions between PINCH1 and WT1 appeared specific, as replacing specific antibodies with control IgG did not result in any binding. Furthermore, physical interaction between endogenous PINCH1 and WT1 was detectable in podocytes after TGF-b1 stimulation, suggesting that PINCH1/WT1 complex formation actually occurs in pathophysiologically relevant conditions. To further confirm the Tasocitinib specificity of PINCH1/WT1 interaction, we employed a GST-fusion protein pull down experiment to examine the interaction between PINCH1 and WT1. To this end, we generated a GST-WT1 fusion protein using a bacterial expression system. As shown in Figure 5D, GST-WT1 fusion protein as well as GST control protein was purified. When these purified proteins were immobilized on glutathione-agarose beads and incubated with podocyte lysates, PINCH1 was pulled down and detected in the assay, indicating a specific interaction between PINCH1 and WT1.