Ab52617

Cells were washed twice in ice-cold PBS and harvested in RIPA buffer (ThermoFisher Scientific) supplemented with protease inhibitors. Cell lysates were centrifuged at 20,000×g for 20 min at 4 ^°C, and the total protein lysate was quantified using Bradford Reagent (B6916; Sigma). Then, 40 μg of total protein lysate was separated on a 4–12% Bis-Tris Plus Gel (Life Technologies) and transferred to a nitrocellulose membrane using the iBlot2 Gel Transfer Device (Life Technologies). Membranes were blocked in 0.5% non-fat dry milk and probed with anti-ABCG1 (1:1000; ab52617, abcam) and antinuclear matrix protein p84 (1:5000; ab487, abcam) primary antibodies anti-rabbit IgG (1:20,000; Ab205718, abcam), and anti-mouse IgG (1:5000; A4416, Sigma Aldrich) secondary antibodies for 1 h at room temperature. Membranes were exposed using Clarity Western ECL Substrate (Bio-Rad), and protein bands were detected on a LI-COR Imaging system with the C-DiGit Image Studio 4.0 software (LI-COR Biosciences, Ltd., UK).

Protein was extracted from cells or tissues using RIPA buffer with protease inhibitors (Sigma). Then, 5 × protein loading buffer was added to the sample at a protein: buffer ratio of 4:1, and the sample was heated in a metal bath at 60 °C for 10 min. Protein electrophoresis was performed using the TGX FastCast acrylamide kit (BIO) and then transferred to PVDF membranes (Millipore, Bedford, MA, America). After blocking in 5% skim milk for 2 h, the membrane was incubated with primary antibody (1:1000 or 1:500) in a refrigerator at 4 °C overnight. GAPDH was used as the loading control. Next, the membrane was washed in (0.1%) (TBS-T) at least three times for 10 min each and incubated with horseradish peroxidation-enhanced secondary antibody (1:10,000) for 2 h. ECL Prime (G.E.) was used to test the protein bands according to the manufacturer's instructions. The antibodies used in the experiment were as follows: GAPDH (ProMab,20,035); ABCA1 (Abcam,ab66217); ABCG1 (Abcam,ab52617); and p-PKCα (Abcam, ab32502;PKCα (Abcam, ab32376).

27-Hydroxycholesterol was provided by American Santa Cruz, and 27-OHC (10 mg) was completely dissolved in a suitable amount of ethanol, then divided equally in centrifuge tubes, with nitrogen gas blowing dry, and finally preserved at −80°C. The 27-OHC working solution contains 0.16% ethanol (v/v). Filipin III was from Cayman Chemical (#70440, Ann Arbor, MI, United States). Primary antibodies for Western blot included mouse anti-ABCA1 (Abcam, ab66217), rabbit anti-ABCG1 (Abcam, ab52617), mouse anti-Caveolin-1 (Abcam, ab17052), rabbit anti-Apolipoprotein E (Abcam, ab52607), mouse anti-LDLR (Millipore, MABS26), rabbit anti-ACAT1 (Abcam, ab168342), rabbit anti-CYP46A1 (Sigma–Aldrich, SAB2100523), mouse anti-KDEL (Santa Cruz, sc-58774), rabbit anti-flotillin (Abcam, ab41927), rabbit anti-SR-B1 (Abcam, 52629), mouse anti- N Cadherin (Abcam, ab98952), mouse anti-Aβ (Abcam, ab126649), and rabbit anti-LRP1 (Abcam, ab92544). Goat anti-mouse (#14709) and rabbit (#7074) biotinylated secondary antibodies were from Cell Signaling Technology. Antibodies for immunofluorescence included anti-CYP46A1 (Abcam, ab82814), goat anti-mouse (Abcam, ab150120), and goat anti-rabbit (Abcam, ab96899).

Eyeballs were harvested from mice that were transcardially perfused with 4% paraformaldehyde and then prepared for RPE–choroid flat mounts or cryosections. The antibodies used for staining were anti-isolectin (FL-1201, 1:500; Vector Labs), anti-F4/80 (ab6640, 1:500; Abcam), anti-SULT2B1 (ab254616, 1:500; Abcam), anti-YM1 (1404, 1:500; Stem Cell), anti-ARG1 (16001-1-AP, 1:500; Proteintech), anti-LXRα (ab176323, 1:100; Abcam), anti-LXRβ (ab28479, 1:200; Abcam), anti-ABCA1 (ab18180, 1:200; Abcam), anti-ABCG1 (ab52617, 1:100; Abcam), and anti-STS (17870-1-AP, 1:500; Proteintech). Images were visualized by a fluorescence microscope (Olympus). ImageJ (National Institutes of Health, Bethesda, MD, USA) was used for analysis and quantification.

The following reagents were used: wortmannin (WMN, 200 nmol/L, MCE); GÖ6983 (4 µmol/L, MCE); API‐2 (1 µmol/L, MCE); mithramycin A (MTA, 200 nmol/L, Enzo); T0901317 (3 µmol/L, Sigma); 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid disodium salt (DIDS, 400 µmol/L, MCE); oxidized low‐density lipoprotein (oxLDL; 50 µg/mL, Yiyuan Biotechnologies); HDL (30 µg/mL, Yiyuan Biotechnologies) and apolipoprotein A‐I (ApoA‐I; 15 µg/mL, Sigma).
The following antibodies were used: anti‐ABCA1 (ab18180, abcam); anti‐ABCG1 (ab52617, abcam); anti‐β‐actin (sc‐47778, santa cruz); anti‐SP1 (NB600‐232, Novus Biologicals); anti‐PKCζ (26899‐1‐AP, proteintech; sc‐17781, santa cruz); anti‐pPKCζ (9378, CST); anti‐α‐SMA (14395‐1‐AP, proteintech); anti‐CD11b (ab184308, abcam); anti‐GADPH (sc‐47724, santa cruz); anti‐SR‐BI (21277‐1‐AP, proteintech); anti‐CD36 (18836‐1‐AP, proteintech) and anti‐SR‐AI (sc‐166184, santa cruz).

The following reagents were purchased from commercial sources: mouse monoclonal anti-human ABCA1 antibody (Ab18180) and rabbit monoclonal anti-human ABCG1 antibody (Ab52617; Abcam, Cambridge, UK); mouse monoclonal anti-human β-actin antibody (A2228) and Oil Red O (O0625; Sigma Aldrich, St. Louis, MO, USA); ProteoExtract Native Membrane Protein Extraction Kit (M-PEK; Calbiochem, Darmstadt, Germany); Quick Start Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA); 7% Tris-acetate gel and TaqMan® Universal PCR Master Mix (Invitrogen, Carlsbad, CA, USA); Mini Protease Inhibitors (Roche, Basel, Switzerland); Immobilon-NC Transfer Membrane (Hybond-C, Amersham International, Little Chalfont, UK); IgG-labeled goat anti-mouse secondary antibody, SP kit, and DAB color reagent (Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd., Beijing, China). The mouse serum, rabbit serum, and all other reagents were from Solarbio (Beijing, China).