Bca detection kit

Proximal jejunum tissue and Caco-2 cells were lysed using RIPA lysate and Phenylmethanesulfonyl fluoride (PMSF; Solarbio Life Sciences, Beijing, China) (100: 1). BCA detection kit (CWBIO, Beijing, China) detects protein concentration. Proteins were then separated on a 10% gel (20μg per lane) using sodium lauryl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Solarbio Co., Beijing, China) (120V, 90minutes). The relevant proteins were then transferred to a 0.45 μm polyvinylidene fluoride membrane (PVDF, Formex, Darmstadt, Germany). Thereafter, the membrane was blocked in Tris buffered saline solution containing 5% skimmed milk powder and 0.1% Tween-20 (TBS-T) at 20° C for 2 h, and then blocked with anti-LXRα, NPC1L1, ABCG5, ABCG8, ACAT2 and β-actin (1: 1000 dilution) (Abcam, Cambridge, UK) was gently shaken at 4° C overnight. The next day, the membrane was rinsed 3 times with TBS-T (10min each time) and incubated with horseradish peroxidase-conjugated secondary antibody (diluted to 1: 5000, Biosharp, Beijing, China) at room temperature for 2 h. Finally, protein bands were visualized by enhanced chemiluminescence (ECL; Merck Millipore, Darmstadt, Germany) and relative protein levels were quantified using Image Lab software.

The hippocampal tissues (20 mg) were collected, homogenized in protein lysate buffer (100–200 μL), and centrifuged at 12,000 rpm for 10 min at 4°C. The total protein concentration was measured using a BCA detection kit (CWBio, Beijing, China). 30–50 μg of protein were separated by 12% SDS‐PAGE and then transferred into polyvinylidene fluoride membranes (Millipore, USA). After blocking with 5% nonfat milk for 2 h at room temperature, the membranes were incubated overnight at 4°C with primary antibodies, including anti‐BDNF antibody (diluted in 1:1000; Abcam, Cambridge, UK), anti‐NGF antibody (diluted in 1:1000; Abcam, Cambridge, UK), and anti‐GAPDH antibody (diluted in 1:2000; Abcam, Cambridge, UK). Then, the membranes were incubated with horseradish peroxidase (HRP)‐coupled secondary antibody (1:5000; Cell Signalling Technology, Hitchin, UK) for 1 h at room temperature. Immunoreactive bands were visualized using ECL‐Plus chemiluminescence reagents (Beyotime Biotechnology, Shanghai, China). The band density was quantified using ImageJ software v1.60 (NIH, Bethesda, USA).