X ray film

Aliquots of homogenate and synaptosomes were lysed in RIPA buffer (50 mM Tris-HCl pH 8.8, 150 mM NaCl, 0.1% SDS, 0.5% NP-40, 0.5% DOC; protease and phosphatase inhibitor cocktail, Sigma-Aldrich) and centrifuged in Eppendorf 5415C microcentrifuge at 14,000 rpm, 5 min, 4°C. Proteins (5 μg/lane, in Sample Buffer: 60 mM Tris-HCl pH 6.8, 10% Glycerol, 2% SDS, 100 mM DTT 0.1% bromophenol blue) were separated in 10%–15% SDS-polyacrylamide gel and transferred to PVDF membranes (Merck-Millipore). Western blot analysis was performed as previously reported (Chun et al., 2004 (link); Volpicelli et al., 2019 (link)). Briefly, filters were blocked for 2 h at RT in 5% (w/v) non-fat milk in Tris-buffered saline Tween-20 (TBST; 0.1% Tween, 150 mM NaCl, 10 mM Tris-HCl, pH 7.5) and probed with the following antibodies: anti-CSTB (1:4,000, ABIN271833 Antibodies), anti-Synaptophysin (SYP; 1:400,000, AB9272, Merck-Millipore), anti-β-actin (1:2,000, 612656, BD Biosciences), and anti-SOD-1 (1:1,000, ADI-SOD-100, Enzo Life Sciences). After several washings in TBST, membranes were incubated with secondary antibody against rabbit (1:20,000, A0545, Sigma-Aldrich) or mouse (1:20,000, NA931, GE Healthcare) linked to horseradish peroxidase. Signals were visualized by chemiluminescence (ECL, Millipore) on autoradiographic film (Fujifilm X-Ray Film).

The methods of protein extraction and Western blot were performed as previously described by Chiu et al. [11 (link)]. Briefly, the radioimmunoprecipitation assay (RIPA) buffer with a cocktail of phosphatase and protease inhibitors (Thermo Fisher Scientific, Waltham, MA, USA) was used for liver protein extraction. A BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to measure protein concentration. For immunoblotting, 50–100 μg of proteins were separated through 8–12% SDS-PAGE gel and then transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). After blocking using 5% non-fat dry milk solution for 1 h, membranes were probed with primary antibodies specific for phosphorylated AMP-activated protein kinase α (p-AMPKα; #2531; 1:1000), AMPKα (#2532; 1:1000) (Cell Signaling Technology, Danvers, MA, USA), PPARγ (sc-7273; 1:1000), and GAPDH (sc-47724; 1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C overnight. The membranes were then probed with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA). An Enhanced Chemiluminescence kit (Bio-Rad) was used to detect the target protein expression, which was exposed to a Fujifilm X-ray film (Tokyo, Japan). The densitometry of target protein was analyzed by the Image J 1.8 software (National Institutes of Health, Bethesda, MD, USA).

The protein expression was measured by Western blotting as described previously [44 (link)]. The equal protein extracts were spiked into 8%–12% sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) gel, and then transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked for 1 h, and then incubated with primary antibodies including AMPKα and phosphorylated AMPKα (p-AMPKα) (Cell Signaling Technology, Danvers, MA, USA), Angptl4, PPAR-γ, SREBP1c, PPAR-α, MTTP, SREBP2, β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and ApoE (Bioss Antibodies, Woburn, MA, USA) overnight. Next, the membranes were hybridized with secondary antibodies. The antigen-antibody complexes were visualized by using Bio-Rad enhanced chemiluminescence kit and exposed to Fujifilm X-ray film (Fujifilm, Tokyo, Japan). The protein bands were densitometrically analyzed with an image software (Image J 1.51; National Institutes of Health, Bethesda, MD, USA).