Ab18180

Mouse liver tissues or macrophages were lysed using RIPA lysis buffer (beyotime) to obtain the protein sample. After the protein concentration was measured using a BCA kit (beyotime), certain volume of protein was mixed with loading buffer (beyotime) for boiling water bath for 3 minutes for degeneration. The proteins were subjected to electrophoresis at 80 V for 30 minutes and then at 120 V for 1–2 hours. Membrane was transferred at a current of 300 mA for 60 minutes and then washed in washing buffer for 1–2 minutes before blocking at room temperature for 1 hou or at 4℃ for overnight. The membranes were incubated with one of the primary antibodies against GAPDH (ab181602, 1:10 000), ApoM (ab85695, 1:1000), FXR1 (ab129089, 1:1000), ABCA1 (ab18180, 1:200), ABCG1 (ab52617, 1:1000) and SR‐BI (ab217318, 1:2000) (Abcam) at shaking bed at room temperature for 1 hour before washing for 3 × 10 minutes. After that, the membranes were incubated with horseradish peroxidase–labelled goat anti‐rabbit IgG (1:5000; Beijing ComWin Biotech Co., Ltd) for 1 hour and washed for 3 × 10 minutes. The membranes were observed under a chemiluminescence imaging system (Bio‐rad) after development solution was added.

Total protein was isolated from peritoneal macrophages using the same procedure described previously [22 (link)]. Protein concentration was measured using a BCA Protein Assay kit (Beyotime Biotechnology, Jiangsu, China). Equal amounts of protein lysates were separated by SDS-PAGE and transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA), followed by blocking with 5% skimmed milk at room temperature for 1 h. Subsequently, the membrane was incubated with the primary antibodies against Abca1 (ab18180, 1:1000; Abcam, Cambridge, MA, USA) and GAPDH (1:2000; Proteintech, Rosemont, IL, USA) at 4 °C overnight. After rinsing with PBS containing Tween for three times, the membrane was incubated with fluorescence-conjugated anti-rabbit IgG secondary antibody (1:10,000; Jackson Immuno Research, West Grove, PA, USA). Western blot bands were obtained by using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA) and quantified with Odyssey v1.2 software (LI-COR Biosciences, Lincoln, NE, USA) by measuring the band intensity (area × OD) in each group and normalizing to GAPDH as an internal control.

The colorectal tissue whole protein was extracted using a protein lysis buffer (P0013B, Beyotime, China). 50 μg of total protein was separated by 10% SDS‐polyacrylamide gel electrophoresis and then transferred onto 0.22 μm polyvinylidene fluoride membrane. Then blocked with 5% nonfat milk powder in TBST for 2 h at room temperature and incubated with the appropriate primary antibody at 4°C overnight. The primary antibodies and their dilution concentration were as follows: anti‐SR‐B1 (1:1000, NB400‐104, Novus Biologicals, USA), anti‐LDL‐R (1:1000, K009497P, Solarbio, China), anti‐PD‐L1 (1:1000, K009918P, Solarbio, China), anti‐GAPDH (1:2500, E12‐052, EnoGene, China), anti‐ABCA1 (1:1000, ab18180, Abcam, UK), anti‐HLA‐B (1:1000, K009916P, Solarbio, China), and anti‐β‐Actin (1:2500, #21338, SAB, USA). Subsequently, the membranes were washed with 1 × TBST and incubated with secondary antibodies: Goat anti‐Mouse or anti‐Rabbit IgG (1:10000) for 1 h at room temperature. After the membranes were washed again by 1 × TBST buffer, the proteins were visualized using enhanced chemiluminescence.

Polyclonal antibodies against Plin1 or Plin2 [7 (link), 8 (link), 10 (link), 16 (link)] were gifts from the laboratory of C. Londos (US National Institutes of Health). This Plin1 antibody was used for immunostaining and another anti-Plin1 antiserum from Abcam (#ab3526) was used for immunoblotting. The sources of other antibodies used were listed as follows: rabbit anti-ATGL from Cayman Chemical (Cat# 10006409), rabbit anti-HSL from Cell Signaling Technology (Cat# 4107s), rat anti-CD36 from (R&D, MAB1955), mouse anti-ABCA1 (Abcam, ab18180), and mouse monoclonal anti-CD68 (Abcam, #ab955),

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).