Chemigenius2

Proteins were extracted from the hippocampus and parietal cortex of the mice, and the content of APP, BACE1, cathepsin B, ADAM10, IDE, Aβ₄₂, p-Tau ser396 and ser404, CDK5, p25/35, PP2A and GFAP were determined by Western blotting as described previously by our laboratory [23 (link),24 (link),25 (link)]. Immobilon western chemiluminescent HRP substrate were used for the visualization of signals and signals were captured through a Syngene chemi-imaging system (Waltham, MA, USA). Subsequently bands were quantified by densitometry via Gene Tool according to the manufacturer’s instructions (SynGene, ChemiGenius2, Perkin Elmer, Waltham, MA, USA). Protein contents of phosphorylated protein were quantified and normalized to the total levels of these proteins. Beta actin was considered as internal controls.

The small intestine tissue samples were lysed in immunoprecipitation lysis buffer (Beyotime, Nantong, China). The lysates were homogenized and centrifuged. The supernatants were collected and the protein concentrations were determined by using a BCA Protein Assay Kit (Beyotime). Equal amounts (30 μg) of ABCA1, ATP-binding cassette G1 (ABCG1), ATP-binding cassette G8 (ABCG8), acetyl-coenzyme A carboxylase (ACC), fatty acid synthase (FAS), low-density lipoproteins receptor (LDLR), LXRα, NPC1L1, PPARα, Sar1B GTPase (Sar1B), scavenger receptor B (SR-B1), SREBP-1, and SREBP-2 were determined by Western blot analysis. Further, the antibodies of ABCA1, ABCG1, ABCG8, ACC, FAS, LDLR, LXRα, NPC1L1, PPARα, Sar1B, SR-B1, SREBP-1, and SREBP-2 were purchased from Abcam (Cambridge, MA, U.S.A.), Cell Signaling Technology (Danvers, MA, U.S.A.), EMD Millipore (Billerica, MA, U.S.A.), or Thermo Fisher Scientific, Inc (Waltham, MA, U.S.A.). Antibody reactivity was detected by Chemiluminescence ECL Detection Systems (EMD Millipore, Billerica, MA, U.S.A.). Subsequently, the intensity of the bands was quantified by densitometry via Gene Tool according to the manufacturer’s instructions (SynGene, Chemi Genius2, PerkinElmer, Wesville, U.S.A.). Beta-actin was used as internal control.