Figure 5.

Increased EGFR- and HGFR-mediated signaling in hepatocytes and regenerating livers of PTP1B^−/− mice. A: The PH was performed in PTP1B^+/+ and PTP1B^−/− mice, and regenerating livers were removed at the indicated periods. Total protein (50 μg) was analyzed by Western blot with the antibodies against phospho-EGFR (pEGFR) (Tyr1086), phospho-HGFR (pHGFR) (Tyr1234/1235), HGFR, phospho-Akt (Akt) (Ser473), Akt, phospho-ERK (pERK) (Thr202/Tyr204), and ERK. Representative autoradiograms of three independent series of PH in each genotype are shown. B: Primary hepatocytes (PTP1B^+/+ and PTP1B^−/−) were serum starved for 4 hours and then stimulated with 20-ng/mL EGF (left) or 30-ng/mL HGF (right) for the indicated periods. Control cells were maintained in the absence of growth factors. At the end of the culture time, cells were lysed and 50 μg of total protein was analyzed by Western blot with the indicated antibodies. Representative autoradiograms are shown. Similar results were obtained in two independent series of experiments. C: Immortalized PTP1B^+/+ hepatocytes were transfected with PTP1B or scramble siRNA oligos. After 48 hours, cells were serum starved for 4 hours and stimulated with 20-ng/mL EGF (left) or 30-ng/mL HGF (right), as described in B, or left untreated. Total protein was analyzed by Western blot with the indicated antibodies. Results shown are representative of two independent experiments. D: Immortalized PTP1B^+/+ and PTP1B^−/− hepatocytes were grown to 60% to 70% confluence and serum deprived for 20 hours. Then, cells were stimulated with TNF-α (10 ng/mL) (T), IL-6 (10 ng/mL) (I), EGF (20 ng/mL) (E), or HGF (30 ng/mL) (H), either alone or in combination, for a further 24 hours (control: C). At the end of the culture time, the percentage of cells in the S phase of the cell cycle was analyzed by flow cytometry, as described in the respective section in Materials and Methods. Results are given as the mean ± SEM (n = 4–8). *P < 0.05 for PTP1B^−/− versus PTP1B^+/+ cells.