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Immunoprecipitation of tyrosine-phosphorylated signaling proteins in chondrocyte lysates. Isolated RZ cells were serum starved for 1 h and then given BDNF (100 ng/ml), CNP (200 ng/ml), or no treatment for 1 h. Cells were then treated with IGF-I (100 ng/ml) for 10 min or no treatment; lysates from treated cells were immunoprecipitated with anti-phosphotyrosine antisera, and the resulting precipitates were analyzed by Western blotting for the presence of IGF-IR, Shc1, or Grb2.
