Insulin receptor substrate (IRS)-1 levels and activation in normal and osteoarthitis (OA) osteoblasts. Cells were grown to confluence and incubated overnight in serum free medium. Cells were treated with 50 ng/ml insulin-like growth factor (IGF)-1 for 5 minutes. (a) After cell lysis in RIPA buffer and immunoprecipitation with anti-IRS-1 antibodies, IRS-1 levels were analyzed by immunoblotting with the same anti-IRS-1 antibody. (b) After cell lysis in RIPA buffer and immunoprecipitation with an anti-IRS-1 antibody, phosphorylated IRS-1 levels were analyzed by immunoblotting with an anti-phosphotyrosine antibody (PY-20). Determination of actin was used as the control for loading. Representative data are shown in panel 1 (a,b). Results expressed as arbitrary scanning units are presented as the mean ± SEM (panel 2 (a,b)).