, then rinsed with 60 isopropanol. Oil Red O staining resolution (1 mL) was added to every well. After incubation for 10 min, the cells were washed thrice with deionized distilled water for the removal of the unbound dye. The stained locations had been imaged and captured applying a Nikon Eclipse Ti2-U and Nikon Eclipse Ts2R camera, respectively (Nikon Co. Ltd., Tokyo, Japan). The quantified levels of captured images have been assessed working with ImageJ software program version 1.52a (National Institutes of Wellness, Bethesda, MD, USA). two.6. Determination of Nuclear Translocation of Peroxisome Proliferator-Activated Receptor (PPAR) Applying Nuclear Fractionation Protein levels of nuclear PPAR and were evaluated by nuclear fractionation. HepG2 cells have been grown in 6-well plates then treated with 20, 40, and 80 TEB or DMSO for 1 and 12 h (n = 3 wells/group). The cells have been washed thrice with PBS and lysed utilizing a hypotonic buffer containing 20 mM Tris (pH 7.4), 10 mM sodium chloride, 3 mM magnesium chloride, and protease inhibitor cocktail (Abbkine, Wuhan, China). The cell lysates have been centrifuged at 600g at 4 C for ten min soon after adding Triton X-100 towards the cell lysates for the extraction of cytosolic proteins. The nuclear proteins have been extracted using a cell extraction buffer containing 100 mM Tris (pH 7.four), 1 mM ethylenediaminetetraacetic acid, two mM sodium orthovanadate, one hundred mM sodium chloride, 1 mM ethylene glycolbis(-aminoethyl ether)-N,N, N N -tetra acetic acid, 10 glycerol, 10 sodium dodecyl sulfate, 1 mM sodium fluoride, 0.five sodium deoxycholate, 1 Triton X-100, 20 mM sodium (1)Foods 2021, ten,4 ofpyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 1 mM aprotinin, and 1 mM leupeptin. Soon after resuspending the pellets working with the cell extraction buffer, the samples have been centrifuged at 14,000g at four C for 30 min. The protein concentrations were then analyzed making use of a bicinchoninic acid protein assay kit, as well as the samples had been utilised for Western blotting. 2.7. Determining Protein Expression Utilizing Western Blotting Protein levels of your lipid metabolism associated cell signaling pathways were measured making use of Western blotting, as described previously [20]. HepG2 cells had been grown in 6-well plates, after which exposed to 20, 40, and 80 TEB or DMSO for 12 and 24 h with or without having NAC pretreatment (five mM in PBS) for 1 h (n = 3 wells/group). The cells were lysed working with a radioimmunoprecipitation assay buffer (Elpis Biotech, Daejeon, Korea). The cell lysates had been then centrifuged at 18,000g at 4 C for 20 min. The protein concentrations have been analyzed making use of a bicinchoninic acid protein assay kit. The protein samples (20 ) had been separated working with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The -separated proteins had been transferred onto a nitrocellulose membrane. BRDT custom synthesis Following that, three nonfat milk buffer was used for blocking the membrane for 1.5 h at 25 C. The membrane was incubated with all the principal antibodies: anti-PPAR (1:1000 dilution; Santa Cruz, CA, USA), anti-PPAR (1:3000 dilution; Cell Signaling Technologies, Beverly, MA, USA), ALK2 supplier anti-cluster of differentiation 36 (CD36) (1:3000 dilution; Cell Signaling Technologies, Beverly, MA, USA), anti-fatty acid transporter protein (FATP) two (1:3000 dilution; Abcam, Cambridge, MA, USA), anti-microsomal triglyceride transfer protein (MTTP) (1:3000 dilution; Abcam, Cambridge, MA, USA), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:20,000 dilution; Merck Millipore, Darmstadt, Germany), and anti-Lamin B (1:5,000 dilution; Santa Cruz
