2005-11-12804A-Z
T. Pawson
Supplemental Methods
Immunoprecipitation and immunoblotting
Lysates were prepared from transfected cells using PLC lysis buffer supplemented with fresh protease inhibitors. Adult rat glomeruli were isolated and processed as described previouslyS1. Following addition of indicated antibodies or GST fusion protein (5 mg), precipitated complexes were resolved by SDS-PAGE. Commercially-available antibodies used were as follows: monoclonal anti-Flag clone M2 (Sigma), monoclonal anti-Myc clone 9E10 (Invitrogen), monoclonal anti-phosphotyrosine clone 4G10, polyclonal anti-Nck1/a (Upstate Biotechnology Inc.), monoclonal anti-phosphotyrosine clone PY69 (BD Pharmingen), monoclonal anti-Nck (BD Pharmingen) and polyclonal anti-GFP 290 (Abcam). Polyclonal antibodies to Nck and Nephrin have been described previouslyS1,S2. Serum recognizing N-WASp was kindly provided by M. Kirschner (Harvard Medical School). Full-length clustering antibodies were kindly provided by K. Tryggvason (Karolinska Institute).
SPOT peptide arrays
High-density peptide arrays (11-mer centered around tyrosine) were synthesized onto acid-hardened cellulose membranes using an AbiMed ASP422 robot with standard Fmoc chemistry (Intavis). OPAL membranes arrayed with the sequence AXXX[pY]XXXXXXA, where X is sequentially replaced by all naturally-occurring amino acids (except for Trp and Cys), were generated as described previouslyS3. Membranes were incubated with 1 mM purified GST fusion protein and HRP-conjugated anti-GST (Santa Cruz Biotechnology) was used to detect bound protein. Alternatively, membranes were incubated with recommended amounts of recombinant Fyn or Src (both Upstate Biotechnology Inc.) for 2 h at room temperature in kinase reaction buffer (50 mM Hepes [pH7.5], 2.5 mM MgCl2, 4 mM MnCl2, 0.1 mM sodium orthovanadate) in the presence of 50 mCi of 32P-gATP (Perkin Elmer) for in vitro kinase assay.
Histological and electron microscopic analysis
For analysis of lacZ expression in Nck1+/- and Nck2+/- mice, kidneys were isolated at P0, fixed in 4% paraformaldehyde/1% glutaraldehyde and stained for b-galactosidase activity using standard techniques followed by paraffin embedding. All other procedures including in situ hybridization analysis and urinalysis were performed as described previously S4.
Supplemental References
S1. Li, H., Lemay, S., Aoudjit, L., Kawachi, H. & Takano, T. SRC-family kinase Fyn phosphorylates the cytoplasmic domain of nephrin and modulates its interaction with podocin. J Am Soc Nephrol 15, 3006-15 (2004).
S2. Bladt, F. et al. The murine Nck SH2/SH3 adaptors are important for the development of mesoderm-derived embryonic structures and for regulating the cellular actin network. Mol Cell Biol 23, 4586-97 (2003).
S3. Rodriguez, M., Li, S. S., Harper, J. W. & Songyang, Z. An oriented peptide array library (OPAL) strategy to study protein-protein interactions. J Biol Chem 279, 8802-7 (2004).
S4. Eremina, V. et al. Glomerular-specific alterations of VEGF-A expression lead to distinct congenital and acquired renal diseases. J Clin Invest 111, 707-16 (2003).
S5. Mundel, P. et al. Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines. Exp Cell Res 236, 248-58 (1997).
S6. Belteki, G. et al. Conditional and inducible transgene expression in mice through the combinatorial use of Cre-mediated recombination and tetracycline induction. Nucleic Acids Res 33, e51 (2005).
S7. Novak, A., Guo, C., Yang, W., Nagy, A. & Lobe, C. G. Z/EG, a double reporter mouse line that expresses enhanced green fluorescent protein upon Cre-mediated excision. Genesis 28, 147-55 (2000).
S8. Klinghoffer, R. A., Sachsenmaier, C., Cooper, J. A. & Soriano, P. Src family kinases are required for integrin but not PDGFR signal transduction. Embo J 18, 2459-71 (1999).
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