The expression of IRS-1 and -2 and the phosphorylation of cellular proteins in cell cultures. (a) Messenger RNA expression patterns of IRS-1 and IRS-2 in osteoblasts and osteoclasts from WT and IRS-1^–/– littermates and mouse osteoclast progenitor C7 cells at various differentiation stages were determined by semiquantitative RT-PCR within an exponential phase of the amplification. Total RNA was extracted from cultured osteoblasts from neonatal calvariae, osteoclasts formed in the coculture of marrow cells and osteoblasts, and C7 cells cultured for 2, 4, and 6 days in the presence of RANKL/ODF and M-CSF. IRS-1 expression was seen only in osteoblasts but not in cells of osteoclastic lineage, even when the amount of template cDNA or the number of amplification cycles was increased. (b) Induction of tyrosine phosphorylation of cellular proteins and IRS-1 in osteoblasts from WT and IRS-1^–/– mice by IGF-I and insulin. Osteoblasts from neonatal calvariae of WT (lanes 1–3, 7–9, 13–15) and IRS-1^–/– (lanes 4–6, 10–12, 16–18) littermates were treated with vehicle (lanes 1, 4, 7, 10, 13, and 16), IGF-I (lanes 2, 5, 8, 11, 14, and 17) or insulin (lanes 3, 6, 9, 12, 15, and 18) for 2 minutes, and extracted cellular proteins were subjected to SDS-PAGE. Lanes 1–6: immunoblotting with an anti-phosphotyrosine antibody; lanes 7–12: immunoblotting with an anti–IRS-1 antibody; lanes 13–18: immunoblotting with an anti–IRS-1 antibody for the osteoblast extracts immunoprecipitated with an anti-phosphotyrosine antibody. Arrowheads indicate 185-kD protein (IRS-1).