Grb2 levels and co-immunoprecipitation with insulin receptor substrate (IRS)-1 in normal and osteoarthitis (OA) osteoblasts. Cells were grown to confluence and incubated overnight in serum free medium. (a) After cell lysis in RIPA buffer Grb2 levels were visualized by immunoblotting with an anti-Grb2 antibody. The data show a representative immunoblot for three normal and three OA osteoblast preparations. (b) In another set of experiments, cells were exposed to 50 ng/ml insulin-like growth factor (IGF)-1 for various length of time or its vehicle, as indicated. After cell lysis in RIPA buffer and immunoprecipitation with an anti-IRS-1 antibody, co-immunoprecipitated Grb2 levels were visualized by immunoblotting with an anti-Grb2 antibody. A representative immunoblotting of four different experiments is presented. Determination of actin was used as the control for loading.