Supplementary Materials Supplementary Data supp_129_1_74__index. effects of iron on the liver proteome were extensive. The top canonical pathway altered by TMHF treatment was the NF-E2Crelated factor 2 (NRF2C)Cmediated oxidative stress response. Because of the long-standing association of elevated hepatic iron with oxidative stress, the remainder of the study was focused on NRF2. TMHF treatment upregulated 25 phase I/II and antioxidant proteins previously categorized as NRF2 target gene products. Immunoblot analyses showed that TMHF treatment increased the levels of glutathione S-transferase (GST) M1, GSTM4, glutamate-cysteine ligase (GCL) catalytic subunit, GCL modifier subunit, glutathione synthetase, glutathione reductase, heme oxygenase 1, epoxide hydrolase 1, and NAD(P)H dehydrogenase quinone 1. Immunofluorescence, carried out to determine the cellular localization of NRF2, showed that NRF2 was detected in the nucleus of hepatocytes from TMHF-treated mice and not from control mice. We conclude that elevated hepatic iron in a mouse model activates NRF2, a key regulator from the mobile response to oxidative tension. Samples were ready based on the Pa State University University of Medication Mass Spectrometry (MS) and Proteomics Primary Facility standard process (http://med.psu.edu/web/core/proteinsmassspectometry/protocols/itraq), adapted through the producers guidelines (Applied Biosystems, Carlsbad, CA). Quickly, liver organ proteins (100 g) from four control and four TMHF-fed mice had been put through acetone precipitation based on the producers guidelines (Applied Biosystems). Examples had been centrifuged at 6000 g for 10min after that, and pellets had been dissolved in 20 l of 0.5M triethylammonium bicarbonate (pH 8.5). Examples were denatured and reduced with the addition of 1 l of 0 in that case.2% SDS and 1 l of 110mM tris-(2-carboxyethyl) phosphine and subsequently incubated at 60C for 1h. One microliter of ready 84mM iodoacetamide was added newly, and samples had been incubated for 30min at space temperature at night. Samples were after that digested with the addition of 10 l (10 g) of Sequencing Quality trypsin (Promega) and incubating over night at 48C. 8-plex iTRAQ reagents had been dissolved in 50 l of isopropanol and individually mixed with each one of the eight tryptic digests, incubated for 2h at space temperatures, quenched by addition of 100 l of drinking water, and pooled. The pooled test was after that dried out and resuspended thrice with 100 l of drinking water. The final resuspension was with 500 l of strong cation exchange (SCX) loading buffer (10mM potassium phosphate, 25% acetonitrile (pH 2.5C3.0). The pooled sample was analyzed by two-dimensional liquid chromatography (LC) separation and matrix-assisted laser desorption and ionization (MALDI) time-of-flight (TOF) tandem mass spectrometry (MS/MS) as described previously (Bortner Peptide identification, protein grouping, and subsequent protein quantitation were done using the Paragon and ProGroup Algorithms (Shilov Liver tissues were homogenized, lysates were prepared, protein concentrations were decided, and gel electrophoresis was carried out as previously described (Moon value of 0.05 was considered statistically significant (Figs. 1B and C). RESULTS Characterization of Livers From Rabbit Polyclonal to eNOS (phospho-Ser615) Mice Fed TMHF C57BL/6 mice were administered a order Lacosamide TMHF-supplemented diet for 1, 2, or 4 weeks or a control diet for 2 or 4 weeks. At the indicated time points, tissue was harvested and subjected to hematoxylin and eosin staining to examine hepatic architecture. Sections from both control and iron-loaded mice exhibited common hepatic architecture, including the presence of mono- and binucleated hepatocytes with circular-shaped nuclei and prominent nucleoli. No histological evidence of cytotoxicity was observed (data not shown) in agreement with our previous data for mice fed a TMHF diet for 4 weeks (Moon = 4). * 0.05, ** 0.001. All changes for mice treated with TMHF were significant in comparison with either 2- or 4-week control. (C, D) Proliferation measured by PCNA positive nuclei. (C) The percentage of PCNA positive cells, as measured by immunohistochemistry, in tissue sections from mice administered a TMHF-supplemented diet for 1, 2, or 4 weeks or a control diet for 2 or 4 weeks was calculated and represented graphically. Data shown are mean SE for each group (= order Lacosamide 4). * 0.05, ** 0.001. All changes for mice treated with TMHF were significant in comparison with either 2- or 4-week control. (D) Representative images of PCNA stained sections from mice fed a TMHF-supplemented diet for 1, 2, or 4 weeks or a control diet for 4 weeks are shown. Arrows indicate PCNA positive nuclei in order Lacosamide hepatocytes in liver sections from TMHF-treated mice. (E) Immunoblot analysis for ferritin, PCNA, and cyclin D1. Protein was extracted from whole livers of mice fed a order Lacosamide TMHF diet for 1, 2, or 4 weeks or control diet for 2 or 4 weeks and subjected to immunoblot analysis for ferritin, PCNA, cyclin D1, or -actin. Stained tissue sections (panels A and D) were photographed by light.