FIGURE 4.

NO-mediated inactivation of endogenous PTPs is important for enhanced insulin responsiveness. A, COS-7 transfectants were stimulated with insulin for 5 min and then harvested at pH 6. 0 in the presence of 1 mm PEO probe. Biotinylated proteins were precipitated by streptavidin paramagnetic particles, eluted, and subjected to immunoblotting with antibodies as indicated (left panel). The right panel shows the results of a densitometric analysis of the gel image as the ratio of biotinylated PTP relative to total PTP in lysate. The data are presented as the means ± S.E. (n = 3). *, p < 0.05, versus insulin-treated mock transfectants. B, COS-7 transfectants were stimulated with insulin for 5 min and harvested in the presence of 15 mm iodoacetic acid. Endogenous PTPs were immunoprecipitated from an aliquot of total lysate (2 mg) and then subjected to in vitro phosphatase activity assay using p-nitrophenyl phosphate as a substrate. The data are presented as the means ± S.E. (n = 3). *, p < 0.05, versus insulin-treated mock transfectants. C, COS-7 transfectants were pretreated with 2 mm sodium orthovanadate for 60 min before insulin stimulation. Immunoprecipitated (IP) insulin receptor and aliquots of total lysate (25 μg) were subjected to immunoblotting with antibodies as indicated (left panel). The right panel shows the results of densitometric analysis of the gel image as the ratio of phosphorylated InRβ relative to total InRβ. A.U., arbitrary unit. Similar results were observed in two independent experiments.