FIG. 3.

Both PTP1B expression and its enzymatic activity were upregulated in the livers of IRS2^−/− mice. A (upper panel): Liver extracts from wild-type (WT), IRS2^−/−, and IRS2^−/−/PTP1B^−/− mice (n = 9 of each genotype) were prepared and total protein (50 μg) was used for Western blot analysis with the corresponding antibodies against insulin-signaling molecules. The anti–β-actin antibody was used as a loading control. A representative experiment is shown. The autoradiograms showing PTP1B from all animals analyzed were quantitated by scanning densitometry. Results are expressed as fold increase of PTP1B content and are means ± SEM. *P < 0.05, IRS2^−/− vs. wild type. Lower panel: Primary hepatocytes obtained from mice of each genotype (wild type, IRS2^−/−, and IRS2^−/−/PTP1B^−/−) were cultured as described in research design and methods. Cell lysates were prepared and total protein (50 μg) was used for Western blot analysis with the corresponding antibodies against PTP1B. A representative experiment corresponding to primary hepatocytes isolated from one animal is shown from three independent experiments performed in triplicate. B: Total RNA was isolated from livers of wild-type and IRS2^−/− mice (n = 9 of each genotype). PTP1B mRNA levels were determined by real-time PCR. Results are expressed as arbitrary units of PTP1B/18S mRNA and are means ± SEM. *P < 0.05, IRS2^−/− vs. wild type. mRNA from IRS2^−/−/PTP1B^−/− mice was used as a negative control. C: PTP1B activity was measured in wild-type and IRS2^−/− liver extracts (n = 5 of each genotype) as described in research design and methods. Results are expressed as pmol · min⁻¹ · μg protein⁻¹ and are means ± SEM. PTP1B activity from IRS2^−/−/PTP1B^−/− liver extracts was used as a negative control. *P < 0.05, IRS2^−/− vs. wild type.