FIGURE 4.

Cell-ECM adhesion and Src activation regulate kindlin-2 tyrosine phosphorylation and kindlin-2·Src complex formation. A and B, cell-ECM adhesion regulates kindlin-2·Src complex formation. Human podocytes were kept in suspension or replated on fibronectin-coated dishes for different periods of time. Kindlin-2 (A) or Src (B) was immunoprecipitated with anti-kindlin-2 or anti-Src antibodies and analyzed by Western blotting with antibodies recognizing kindlin-2 or Src. C, human podocytes were treated with or without H₂O₂ for 15 min, and the cell lysates (lanes 1 and 2) were analyzed by Western blotting with antibodies recognizing Src, phospho-Src (Tyr-416), or GAPDH. Kindlin-2 tyrosine phosphorylation and complex formation with Src were analyzed by co-immunoprecipitation (lanes 5–7). The cell lysates (lanes 3 and 4), anti-kindlin-2 immunoprecipitates (lanes 6 and 7), or control IgG immunoprecipitates (lane 5) were analyzed by Western blotting with antibodies recognizing phosphotyrosine, kindlin-2, or Src as indicated. D, human podocytes were pretreated with (lanes 2 and 4) or without (lanes 1, 3, and 5) PP2 and then treated with H₂O₂ for 15 min. The cell lysates (lanes 1 and 2), anti-kindlin-2 immunoprecipitates (lanes 3 and 4), or control IgG immunoprecipitates (lane 5) were analyzed by Western blotting with antibodies recognizing phosphotyrosine, kindlin-2, or Src. E, the lysates (lanes 1 and 4), anti-kindlin-2 immunoprecipitates (lanes 3 and 6), or control IgG immunoprecipitates (lanes 2 and 5) derived from human podocytes expressing wild type Src (lanes 1–3) or SrcY527F (lanes 4–6) were analyzed by Western blotting with antibodies recognizing phosphotyrosine, kindlin-2, or Src. F, human podocytes were transfected with vectors encoding SrcY527F or control vectors and treated with or without PP2, as indicated. The cell lysates (lanes 1–3), anti-kindlin-2 immunoprecipitates (lanes 5–7), or control IgG immunoprecipitates (lane 4) were analyzed by Western blotting with antibodies recognizing phosphotyrosine (P-Tyr-100), kindlin-2, and Src. *, position of a nonspecific band derived from the anti-kindlin-2 antibody recognized by P-Tyr-100. G, the lysates (lanes 1 and 4), anti-kindlin-2 immunoprecipitates (lanes 3 and 6), or control IgG immunoprecipitates (lanes 2 and 5) derived from human podocytes expressing SrcY527F (lanes 1–3) or SrcK295M/Y527F (lanes 4–6) were analyzed by Western blotting with antibodies recognizing phosphotyrosine, kindlin-2, or Src.