Srebp1

For protein immunoprecipitation, cells (1 × 10⁷) overexpressed Flag-tagged NUPR1 were harvested, and lysate samples containing 2000 μg total protein were immunoprecipitated with 4 μl M2-Flag (Sigma) or SREBP1 (Santa Cruz Biotechnology) respectively at 4 °C overnight, followed by incubation with 40 μl Protein A/G plus agarose beads (Santa Cruz Biotechnology) at 4 °C for 4 h. Beads were washed and boiled in 40 µl of loading buffer, and then western blotting was conducted using SREBP1 (Proteintech, 14088-1-AP) or anti-NUPR1 (Proteintech, 15056-1-AP). The protein of input used in Fig. 4-A was about 280 µg and 8 μl used for IP.

As described previously with modifications [28 ], the aHSCs were lysed in a lysis buffer (Sigma-Aldrich, Missouri, USA). The lysed cells were centrifuged at 15,000 rpm for 15 min at 4°C; then, the supernatants (30 μg protein) were loaded to SDS-PAGE and transferred onto a PVDF membrane. The membrane was blocked using 1% BSA in PBS containing 0.1% Tween-20 for 1 h at room temperature. The proteins were detected through an overnight incubation at 4°C with primary antibodies against a-SMA (mouse monoclonal), β-actin (mouse monoclonal) (Sigma-Aldrich, Missouri, USA), collagen I (mouse monoclonal), cyclin D-1 (rabbit polyclonal), p27 (rabbit polyclonal), SREBP1 (mouse monoclonal) (Santa Cruz Biotechnology, CA), and PPARγ (rabbit monoclonal) (Cell Signaling Technology, Beverly, MA). Finally, the membrane was incubated with a HRP-conjugated secondary antibody for 1 h. An enhanced chemiluminescence kit (Millipore, Bedford, MA) was used to visualize the reactive signals, quantified by ImageJ software.

Liver tissues from each mouse were homogenized at 4 °C in extraction buffer (100 mM Tris-HCl, pH 7.4, 5 mM EDTA, 50 mM NaCl, 50 mM sodium pyrophosphate, 50 mM NaF, 100 mM orthovanadate, 1% Triton X-100, 1 mM phenylmethanosulfonylfluoride, 2 mg/mL aprotinin, 1 mg/mL pepstatin A, and 1 mg/mL leupeptin) and centrifuged at 3000×g for 15 min at 4 °C. HepG2-HBx cells were lysed in 250 μL of sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM dithiothreitol, and 0.1% bromophenol blue). The amount of proteins in the tissue lysate and cell lytsate were assayed using the Bradford assay. Proteins (40 μg/sample for homogenized tissue, 10 μg/sample for cell lysates) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and protein bands were transferred electrophoretically to nitrocellulose membranes. Membranes were probed with polyclonal antibodies against SREBP1 (Santa Cruz Biotechnology, Santa Cruz, CA), GFP, AKT, AKT-p, PI3K, PI3K-p, C/EBPα, and β-actin (as a loading control) (Cell Signaling Technology, Beverly, MA, USA). Bound antibodies were detected using peroxidase-conjugated anti-rabbit antibodies followed by chemiluminescence assay (ECL System; Amersham, Buckinghamshire, UK) and autoradiographic exposure. The intensity of band on the bolts was calculated by Gel-Pro® Analyzer (Media Cybernetics, Inc., Silver Spring, MD).

The livers of 3-week-old male offspring were homogenized in RIPA buffer (Biosesang, Seongnam, Korea) containing a protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany), and the supernatants were collected by centrifugation (16,600× g for 15 min at 4 °C) (n = 9). Protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Rockford, IL, USA). Samples containing 20 μg protein were applied to an 8% (w/v) sodium dodecylsulphate–polyacrylamide gel. After electrophoresis, the proteins were transferred to nitrocellulose membranes (Whatman, Dassel, Germany) and probed with primary antibodies against AKT, AMPKα, and ACCα (Cell Signalling Technology, Inc., Boston, MA, USA). mTOR was purchased from Merck Millipore (County Cork, Ireland). SREBP1, ChREBP, FAS, and beta-Actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). beta-Actin served as the reference protein for normalization of band intensity. All bands were visualized using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).

Total liver extracts were prepared as for MTHFR assays (see previous paragraph). Nuclear and cytoplasmic fractions were isolated by using the NE-PER Nuclear Protein Extraction Kit (Thermo Scientific) following the manufacturer’s instructions. Protein concentration was determined by Bradford assay by using bovine serum albumin as a standard. Western blotting was performed with conventional methods by using 25 μg total or cytoplasmic protein or 15 μg nuclear protein. Primary antibodies were sterol regulatory element-binding protein (SREBP-1; Santa Cruz Biotechnology), cyclic AMP-responsive element-binding protein (Cell Signaling Technology), β-actin (Sigma-Aldrich), and MTHFR (35 (link)). Secondary antibody was horseradish peroxidase–coupled anti–rabbit IgG (GE Healthcare). Bands were visualized with Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare) and film exposure and quantified by densitometry by using Quantity One 1-D Analysis Software version 4.6.9 (Bio-Rad). Results were normalized to the β-actin loading control and reported relative to the mean value for CD-fed Mthfr^+/+ mice, which was standardized to a reference value of 1.