Fixed mouse liver

tissues were embedded Selleckchem U0126 in paraffin and 5-μm sections were used for immunohistochemistry. Cell proliferation was assessed by way of KI-67 staining as described.19 KI-67–positive cells were quantified by counting hepatocytes in at least 10 random fields. Sections from 2 to 4 individual animals for each genotype were used, and the data are expressed as the mean ± standard deviation (SD). For Western blot analysis, livers were lysed at the indicated time points after PH in lysis buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 1 mM ethylene diamine tetraacetic acid, 10% glycerin, 1% Triton X-100, 10 mM Na4P2O7) and protein concentrations were measured with the BCA Assay. Nitrocellulose membranes were blocked in either 1× NET Gelatine or 5% milk/TBS-T and processed for immunoblotting. Primary antibodies against mig-6 (9; cl. PE-16), Tubulin, and β-actin were from Sigma; p-EGFR Y1173, EGFR, p-c-Jun Ser66, and pan-phospho-tyrosine antibodies (cl. Androgen Receptor antagonist 4G10) were from Upstate; p-ERK1/2, p-Rb and p-AKT Ser473 were from Cell Signaling Technologies. Secondary horseradish peroxidase–conjugated anti-mouse (Sigma), anti-rabbit (BioRad), and anti-sheep (Jackson ImmunoLaboratories) antibodies were used at a 1:10,000 dilution and detected using an ECL reagent

(Perkin Elmer). For immunoprecipitation, cell lysates were prepared in HNTG buffer (20 mM Hepes [pH 7.5], 150 mM NaCl, 0.1% Triton X-100, 10% glycerin, and 10 mM Na4P2O7), followed by incubation overnight at 4°C with 40 μL of protein A beads and 2 μg/mL anti-EGFR (homemade; cl.108.1) or anti-mig-6 antibody (homemade; cl. 1575). Total RNA was isolated from fresh liver tissue using Trizol (Sigma), and complementary DNA was synthesized using AMV Reverse Transcriptase (Roche). Reverse-transcription polymerase chain reaction primers were as follows: EGFR, 5′-GGAGGAAAAGAAAGTCTGCC-3′ (forward) and 5′-ATCGCACAGCACCAATCAGG-3′ (reverse) (Tm = 53°C, 28 cycles); HB-EGF, 5′-GCTGCCGTCGGTGATGCTGAAGC-3′ (forward) and 5′-GATGACAAGAAGACAGACG-3′ (reverse) (Tm = 60°C; 28 cycles); TGFα, 5′-CTCTGCTAGCGCTGGGTATCC-3′ PRKACG (forward) and 5′-AGGGCGCTGGGCTTCTCAT-3′

(reverse) (Tm = 58°C; 28 cycles); and cyclophilin A, 5′-GACGCCACTGTCGCTTTTCG-3′ (forward) and 5′-CTTGCCATCCAGCCATTCAGTC-3′ (reverse) (57°C; 24 cycles). All polymerase chain reactions were performed with an Eppendorf thermocycler. Hs 817.T and HepG2 human liver cancer cell lines were purchased from the American Type Culture Collection and cultured according to its guidelines. Primary hepatocytes were cultured on collagen I-coated (BD Biosciences) cell culture dishes in William’s E medium (Invitrogen, CA) containing 10% fetal bovine serum. All cells were starved in medium containing 0.1% fetal bovine serum overnight, stimulated with 50 ng/mL EGF (Gibco), and lysed at the indicated time points, and protein lysates were subjected to western blot analysis or immunoprecipitation. Phase contrast pictures were taken on a Zeiss Axiovert 300 microscope.