PTP1B binds to and dephosphorylates nephrin. (a) Cos-1 cells were transiently transfected with nephrin and GST-PTP1B-WT or GST-PTP1B-DA (substrate-trapping mutant). Cell lysates were subjected to pull-down (PD) with glutathione beads and analyzed by immunoblotting. PTP1B-DA, but not WT, pulled down nephrin while preserving its phosphorylation. Nephrin pulled down by PTP1B-DA appeared as a doublet and both bands were phosphorylated—the bottom band corresponded to nephrin at 180 kDa, while the top band (indicated by *) ran above 180 kDa, possibly representing a hyperphosphorylated form. (b) Pull-down by PTP1B-DA was performed using Y-to-F mutants of nephrin. Mutation of the two Nck binding sites in rat (Y1204 and Y1228) [5] additively affected nephrin-PTP1B interaction, while mutation of another Y residue (Y1152, phosphoinositide-3 kinase binding site) [8] had no effect. Densitometric analysis, normalized to total nephrin, is shown. * versus WT, . (c) Undifferentiated cultured mouse podocytes were serum starved in 0.5% FBS overnight and were incubated with the PTP1B inhibitor (50 μM) or vehicle for the indicated times. Cell lysates were analyzed by immunoblotting for pY1217h (=pY1228r)-nephrin and total nephrin. Bottom: densitometric analysis is shown. Results were normalized to total nephrin. * versus no inhibitor, .