FIG. 6.

Characterization of WT and IRS-2-null mammary tumor cell lines. (A) Aliquots of cell extracts from WT, IRS-2^−/−, and IRS-1^−/− mammary tumor cells that contained equivalent amounts of total protein were immunoblotted with antibodies specific for IRS-1, IRS-2, the β4-integrin subunit, and the p85 regulatory subunit of PI3K as a loading control. PyV-MT mammary tumor cells were stimulated for 10 min with IGF-1 (100 ng/ml), and aliquots of cell extracts that contained equivalent amounts of total protein were immunoprecipitated with antisera specific for the IGF-1R β subunit. The immunoprecipitates were immunoblotted with phosphotyrosine-specific antibodies (pIGF-1Rβ). The immunoblots were subsequently stripped and reprobed with IGF-1R β-subunit-specific antisera (IGF-1R). Two independent subclones for each tumor genotype are shown. (B) H&E-stained section from an IRS-2^−/− tumor grown orthotopically in the mammary fat pad of a WT mouse. The tumors are solid, with little glandular structure, and are high grade. (C) CD31 staining of an IRS-2^−/− tumor grown orthotopically in the mammary fat pad of a WT mouse. WT, mice that are homozygous positive for the IRS-2 gene; IRS-2^−/−, mice that are homozygous null for the IRS-2 gene; IRS-1^−/−, mice that are homozygous null for the IRS-1 gene.