FIGURE 3.

Identification of PLC-γ1 as a phospho-Nephrin-binding protein. A, recombinant GST-Nephrin-CD was bound to glutathione-Sepharose beads and incubated with recombinant Fyn with or without ATP. After washing, the beads were incubated with lysates from HEK293T cells, and bound proteins were analyzed by silver staining. These experiments were repeated four times, and representative data are shown. B, multiple proteins specifically bind to phosphorylated Nephrin. Protein samples in A were analyzed by immunoblotting for the indicated antibodies. C, expression of PLC-γ1 in podocytes. Indirect immunofluorescence microscopy was performed to detect PLC-γ1 in adult rat kidney cryosections (left). Colocalization with vimentin suggests that PLC-γ1 is highly expressed in podocyte in renal glomeruli (right). Magnification was ×400.