Fatty acid synthase fas

Protein from tissues was extracted with Pro-Orep (iNtRON Bio, Seongnam, Korea) according to the manufacturer’s instructions. Western blot analysis was performed with protein lysates using antibodies to sterol regulatory element-binding protein 1 (SREBP1; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and phospho-NF-κB, total NF-κB, phospho-IκBα, total IκBα, fatty acid synthase (FAS), and GAPDH (all from Cell Signaling Technologies, Danvers, MA, USA).

Western blot was performed as previously reported [14 ]. The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Gendepot). The membrane was incubated following primary antibodies: p-AMP-activated protein kinase (AMPK), AMPK (Cell signaling, Beverly, MA, USA), sterol regulatory-element binding proteins (SREBP)1c (Santa Cruz Biotechnology, Dallas, TX, USA), fatty acid synthase (FAS; Cell signaling), peroxisome proliferator-activated receptor-γ (PPARγ; Cell signaling), CCAAT/enhancer-binding protein-α (C/EBPα; Cell signaling), SREBP1 (Abcam), SREBP2 (ABclonal, Wuhan, Hubei, China), cytochrome P450 family 7 subfamily a member 1 (CYP7A1; ABclonal), 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMCGR; Santa Cruz Biotechnology), β-actin (Santa Cruz Biotechnology), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology). The secondary antibodies, including Goat anti-Mouse IgG (H+L) and Goat anti-Rabbit IgG(H+L)-HRP, were obtained from Gen-depot. The blots were visualized using the West-Q Femto Clean enhanced chemiluminescence (ECL) solution (Gendepot) and LuminoGraph III Lite (ATTO, Tokyo, Japan), following the manufacturer’s instructions. Quantitative analysis was measured with CS Analyzer 4 (ATTO).

Formalin-fixed, paraffin-embedded, de-waxed sections 4 µm thick of the frontal cortex (GM) and subcortical WM in five MA cases were processed for specific immune-histochemistry. The sections were boiled in citrate buffer (20 min) to retrieve protein antigenicity. Endogenous peroxidases were blocked by incubation in 10% methanol-1% H₂ O₂ solution (15 min) followed by 3% normal horse serum solution. Then the sections were incubated at 4ºC overnight with one of the primary rabbit polyclonal antibodies: 3-ketoacyl-CoA thiolase (ACCA1) (MyBioSource MBS1492126) used at a dilution of 1/100; Fatty Acid Synthase (FAS) (C20G5, Cell Signaling 3180) diluted 1/50, and Stearoyl-CoA desaturase (SCD) (MyBioSource, BS421254) used at a dilution of 1/50. After incubation with the primary antibody, the sections were incubated with EnVision+ system peroxidase (Dako, Agilent Technologies, Santa Clara, CA, USA) for 30 min at room temperature. The peroxidase reaction was visualized with diaminobenzidine and H₂ O₂. Control of the immunostaining included omission of the primary antibody; no signal was obtained following incubation with only the secondary antibody. Sections were slightly counterstained with hematoxylin. Due to the individual variability of the immunostaining, no attempt at quantification was performed; immunohistochemistry was used to assess the localization of the enzymes.

The proteins were extracted from the fully differentiated 3T3-L1 cells using passive lysis buffer (Promega Corporation, Fitchburg, WI, USA) supplemented with a proteinase inhibitor (MedChemExpress, Monmouth Junction, NJ, USA) and phosphatase inhibitor (MedChemExpress), referring to the manufacturer's descriptions, and were quantified by BCA assay kit (Life Technologies). The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with the indicated antibodies: peroxisome proliferator-activated receptor gamma (PPARγ; dilution 1 : 1000; Cell Signaling Technology, Beverly, MA, USA), CCAAT/enhancer-binding protein alpha (C/EBPα; dilution 1 : 1000; Cell Signaling Technology), fatty acid-binding protein 4 (FABP4; dilution 1 : 1000; Cell Signaling Technology), fatty acid synthase (FAS; dilution 1 : 1000; Cell Signaling Technology), protein kinase B (AKT; dilution 1 : 1000; Bioss, Woburn, MA, USA), phospho-Akt (p-AKT; dilution 1 : 2000; Cell Signaling Technology), tumor necrosis factor-alpha (TNF-α; dilution 1 : 1000; Cusabio Life Science, Wuhan, China), and GAPDH (dilution 1 : 2000; BioLegend, San Diego, CA, USA). GAPDH was used to achieve equal loading of protein. The protein signals on the membranes were developed using ECL western blotting substrate (Daeil Lab Service).

3T3L-1 cells were lysed in a buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ethylene glycol tetraacetic acid (EGTA), and 1% (v/v) Nonidet P-40 (NP-40). A protease inhibitor cocktail (Roche) and phosphatase inhibitors (10 mM phenylmethylsulfonyl fluoride, 1% sodium pyrophosphate, 10 mM sodium fluoride, and 2 mM sodium vanadate) were added. By SDS-polyacrylamide gel electrophoresis (PAGE), 20 µg proteins were separated, then transferred to PVDF membranes (Millipore, Burlington, MA, USA) for immunoblotting. Membranes were probed with antibodies for PPARγ (#2435), C/EBPα (#8178), perilipin (#9349), fatty acid synthase (FAS) (#3180), acetyl-CoA carboxylase (ACC) (#3676) and caspase 3 (#9662) (Cell Signaling, Danvers, MA, USA). Protein concentration was measured with Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Walthom, MA, USA). As control, β-actin (sc-47778) (Santa Cruz, CA, USA) was used for normalization. Band intensities were quantified using ImageJ (http://rsbweb.nih.gov/ij).

Freeze-clamped liver tissues were homogenized in ice-cold RIPA lysis buffer at pH 7.5 supplemented with protease and phosphatase inhibitor cocktails. Protein levels in tissue homogenates were determined using the bi-cinchonnic acid method (Bio-Rad Laboratories Inc., USA). Liver lysates containing equal amount of proteins were resolved by SDS-PAGE. Activation of key insulin signaling proteins and levels of lipogenic and gluconeogenic enzymes were examined by immunoblotting using specific antibodies. Forkhead box protein O1 (FoxO1), p-FoxO1^Ser256, HDAC4, p-HDAC4^Ser632, HDAC5, p-HDAC5^Ser498, HDAC1, AMPK, p-AMPK^Thr172, acetyl-CoA carboxylase (ACC), p-ACC^Ser79, fatty acid synthase (FAS), stearoyl-CoA desaturase 1 (SCD1), Akt, p-Akt^Ser473, glycogen synthase kinase 3β (GSK3β) and p-GSK3β^Ser9 were obtained from Cell Signaling (USA). Acetyl-FoxO1^{lys259, 262 and 271} and sterol regulatory element-binding protein 1c (SREBP-1c) were purchased from Santa Cruz (USA). HAT1 and sirtuin 1 (SIRT1) were from Abcam and Millipore (USA) respectively. Quantitative densitometry analysis was performed using Image Lab software (Bio-Rad Laboratories Inc., USA).