Bs 5013r

Rosmarinic acid (536954), metformin hydrochloride (PHR1084), 2-mercaptoethanol (M3148-25ML), protease inhibitor cocktail (P8340-5ML), and antibody against β-actin (a2066) were purchased from Sigma-Aldrich (St. Louis, MO, USA). A mouse IL-1β ELISA kit (ab100704) and antibodies against ABCA1 (ab18180), LDL-R (ab52818), and carnitine palmitoyltransferase 1A (CPT1A; ab53532) were purchased from Abcam (Cambridge, UK). Antibodies against p-AMPKα^Thr172 (#2531) and AMPK (#2532) were purchased from Cell Signaling Technology (Beverly, MA, USA). An antibody against ABCG5 (bs-5013R) was obtained from Bioss Antibodies (Woburn, MA, USA). Antibodies against ABCG8 (NBP1-71706) and SR-BI (NB400-104) were procured from Novus Biologicals (Littleton, CO, USA). Anti-cholesterol 7 alpha-hydroxylase A1 (CYP7A1) antibody (PA5-100892) was purchased from Invitrogen (Waltham, MA, USA). Accu check (code 222) was obtained from Roche Diagnostics (Mannheim, Germany). A cholesterol fluorometric assay kit (10007640) and triglyceride colorimetric assay kit (10010303) were procured from Cayman Chemical (Ann Arbor, MI, USA).

About 40 mg of frozen liver and the entire middle segment of small intestine that was divided into 5 equal segments (~40 mg) were homogenized in RIPA buffer (50 mMTris-HCl, pH 8.0, 150 mMNaCl, 2 mM MgCl₂, 0.1% SDS, 1.5% Nonidet P-40, and 0.5% deoxycholate) with protease inhibitors. The homogenate was centrifuged at 12,000 rpm for 20 min at 4 ^oC. The total protein concentration of each sample was determined by a BCA protein assay kit (Pierce, Rockford, USA). From each sample, a total of 50 μg of total proteins was subjected to SDS-PAGE (8% for NPC1L1, 6% for ABCA1 and 10% for others) and Western blotting. Primary antibodies included a rabbit anti-mouse CYP7A1 antibody (Cat.#: ab65596, Abcam, UK), a rabbit anti-mouse/human LDL receptor antibody (Cat.#: ab52818, Abcam, UK), a mouse anti-ABCA1 antibody (Cat.#: ab18180, Abcam, UK), a rabbit anti-SR-BI antibody (Cat.#: ab52629, Abcam, UK), a rabbit anti-ABCG5 antibody (Cat.#: BS5013R, Bioss, China), and a goat anti-NPC1L1 antibody (Cat.#: ab121000, Abcam, UK).