Additionally, our in vitro studies provide the first evidence that epidermal growth factor (EGF)-mediated proliferation is critically dependent on eNOS expressed in hepatocytes. AP-1, activator protein-1; BrdU, 5′-bromo-2′-deoxyuridine; cDNA, complementary DNA; ECM, extracellular matrix; EDTA, ethylenediaminetetraacetic acid; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; Egr-1, early growth response-1; EMSA, electrophoretic mobility-shift assay; eNOS; endothelial nitric oxide synthase; ERK, extracellular signal-regulated

kinase; HGF, hepatocyte growth factor; iNOS, inducible nitric oxide synthase; MAPK, mitogen-activated protein kinase; MMP-9, matrix metalloprotease-9; mRNA, messenger RNA; NO, nitric oxide; P13K, P13 Y-27632 price kinase; PCNA, proliferating cell nuclear antigen; PH, partial hepatectomy; qRT-PCR, quantitative reverse-transcription polymerase chain reaction; SD, standard deviation; TUNEL, terminal

deoxynucleotidyltransferase-mediated UTP nick-end labeling; VEGF, vascular endothelial growth factor; WT, wild type. C57BL6/J wild-type (WT) mice and eNOS−/− mice in C57BL6/J background were purchased from Jackson Laboratories (Bar Harbor, ME). Mice were housed in a temperature-controlled animal facility with 12-hour light-dark cycles and were maintained on a standard diet and water. All experiments Ceritinib purchase were conducted in accord with the National Institutes of Health (Bethesda, MD) Guidelines for the Care selleck chemical and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the Baylor College of Medicine (Houston, TX). WT and eNOS−/− adult male mice (10-12 weeks) were subjected to

70% PH under light isoflurane anesthesia in the midmorning, based on the method described by Higgins and Anderson.13 Briefly, the left lateral and median lobes were individually ligated and excised, then the right lateral and caudate lobes (i.e., remnant livers) were harvested at various time points (5 minutes to 8 days). Sham operation consisted of laparotomy and mobilization of the liver without the ligation or resection of liver lobes. Total protein extracts were obtained by homogenizing liver tissues in total lysis buffer (50 mM of Tris-HCl, pH 7.5, 0.5 M of NaCl, 2 mM of ethylenediaminetetraacetic acid [EDTA], 2 mM of ethylene glycol tetraacetic acid, 1.0% Triton X-100, 0.25% deoxycholate, 1.0 mM of phenylmethylsulfonyl fluoride, 1 μg/mL of pepstatin, 1.0 μg/mL of leupeptin, 1.0 μg/mL of aprotinin, 2.0 mM of NaF, and 2.0 mM of activated Na3VO4) and centrifuging at 14,000 rpm for 10 minutes. Nuclear protein extracts were prepared as described previously.14 Briefly, liver tissues were gently homogenized in lysis buffer (25-mM Tris buffer, pH 7.5, containing 0.5 mM of EDTA, 1.5 mM of MgCl2, 420 mM of NaCl, and protease inhibitors).