Ab30532

Cells grown to near confluence on 6 cm-dishes were lysed, separated by SDS gel electrophoresis and blotted onto polyvinylidene difluoride membranes. Antibodies directed against Abca1 (ab18180), LDL receptor (ab30532), HMG-CoA reductase (ab174830), SREBP-2 active fragment (ab30682), NPC1 (ab134113), and APP (ab32136) were obtained from Abcam (Cambridge, UK). Antibodies to AMPKα (#2603 S) and phospho-Thr172-AMPKα (#2535 S) were from Cell Signaling Technology (Danvers, MA, USA), while anti-β-actin (A5441) was from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). HRP-conjugated secondary antibodies were from GE Healthcare (Freiburg, Germany). The enhanced chemiluminescence system was from Millipore Corporation (Billerica, MA, USA).

Cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, blocked with 5% BSA, and then incubated with the LDLR antibody (ab30532, Abcam) overnight at 4 °C. The cells were then washed, incubated with the secondary antibody (goat anti-rabbit IgG Alexa488) for 1 h at room temperature, and stained with Hoechst for cell nuclei. Confocal images were captured with the Zeiss LSM 880 NLO with AiryScan System. Colocalization of Tcnα and LDLR was analyzed by the software ImageJ ver1.53.

The sections were repaired with an EDTA antigen repair buffer (pH = 8, G1206, Servicebio, CHN) in a microwave, and the 3% BSA was added dropwise to block for 30 min. Then the CD45R (1 : 50, sc19597, Santa, US), INSR-β antibody (1 : 50, sc57342, Santa, US), CXCL16 (1 : 100, DF13312, Affinity, CHN), anti-LDL receptor antibody (1 : 100, ab30532, Abcam, US) and anti-TF antibody (F-8) (1 : 50, sc-373785, SCBS, US) were used for incubating with the sections overnight at 4°C, and then after washing, the tissue that was incubated with the goat antirabbit IgG H&L (ab150078, Abcam, US) that was incubated in dark for 50 min. Following PBS washing, the nuclei were counterstained with DAPI (G1012, ServiceBio, CHN). Thirdly, after being washed, the sections were quenched by the antifluorescence quenching sealing reagent (G1401, Servicebio, CHN) for 5 min and rinsed with running water for 10 min. Between every two steps, the sections were washed 3 times with PBS (pH = 7.4) on the shaking table. Finally, a NIKON eclipse upright microscope was used to observe and image the fluorescence (the nucleus is blue, and the positive expression is red or green).

The 786-O, ACHN and HK2 were purchased from the Chinese Academy of Sciences cell bank. All cells were cultured in the presence of penicillin/streptomycin at 37 °C in air containing 5% CO₂. The antibodies used included mouse anti-ABCA1 (Abcam, ab18180), rabbit anti-LDLR (Abcam, ab30532), rabbit anti-HMGCR (Abcam, ab174830), rabbit anti-SREBP-1c (Abcam, ab28481), mouse anti-SCD1 (Abcam, ab19862), rabbit anti-SRB1 (Abcam, ab217318), rabbit anti-FASN (CST, 3180), rabbit anti-Bax (Abcam, ab32503), rabbit anti-Bcl2 (Abcam, ab59348), rabbit anti-Caspase3 (Abcam, ab32042), and anti-HMGCR (used for IHC, Proteintech, 13533). Cholesterol-methyl-β-cyclodextrin (Sigma, C4951), SR9243 (MCE, 16972), LXR623 (MCE 10629), stearic acid (MCE, B2219), oleic acid (MCE, N1446) and palmitic acid (MCE, N0830) were also utilised.

Frozen rat liver was homogenized in 20 mM Tris–HCl buffer (pH 7.8) containing 0.2% Triton X-100 and protease inhibitor cocktail (Sigma, USA), and then centrifuged (15 000 × g, 20 min, 20 °C). Aliquots of the obtained supernatants containing 10 µg of protein were separated by 10% SDS-PAGE and electroblotted to Immuno-Blot™ PVDF Membrane (Bio-Rad Laboratories, Hercules CA, USA). The membrane was blocked by incubation with blocking buffer, and then incubated with rabbit polyclonal anti- HNF4α antibody (NBP1-00876, Novusbio), mouse monoclonal anti-HNF1α antibody (GTX12064, GeneTex), rabbit polyclonal anti- LDL-Receptor antibody (AB30532, ABCAM), goat polyclonal anti-PCSK9 (AF3985-SP, R&D Systems), and rabbit polyclonal anti-actin antibody (A 5060, Sigma–Aldrich). Secondary HRP-conjugated antibodies were obtained from Sigma Aldrich (A0545, A9044, A5420). The reactions were visualized with a SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific Inc., Rockford, IL, USA). The bands (visible on the film after the chemiluminescent detection) were compared to molecular mass protein markers (SM1811) obtained from Fermentas, visible on the membrane after electroblotting. The film was adjusted to the membrane in such way that the membrane edges were visible on the film. Blots were analyzed using Quantity One program, version 4,0 (Bio-Rad).

Cells were lysed in RIPA buffer [10 mmol/l Tris (pH 7.4), 150 mmol/l NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, and complete EDTA (Roche)] with protease inhibitors (Roche). Equal amounts of protein were separated on SDS-PAGE and trans-blotted onto PVDF membrane (GE Healthcare). Membranes were blocked in appropriate blocking buffer recommended for the antibody (TBS-T supplemented with 5% milk) and incubated overnight on a shaker at 4°C with primary antibodies in the same blocking buffer. Membranes were incubated for 1 h with a HRP-conjugated secondary antibody (Dako) in the blocking buffer. Membranes were further incubated with chemiluminescence substrate for 1 min (Pierce ECL Plus; Thermo Scientific) and imaged using Fusion Fx (Vilber). The expression of LDLR (1:1,000, ab30532; Abcam), VLDLR (1:1,000, NBP1-78162; Novus), SR-BI (1:1,000, NB400-131; Novus), and NRP1 (1:1,000, ab81321; Abcam) were evaluated and compared with the expression of TATA binding protein (TBP) (1:1,000, ab51841; Abcam), which was used as a loading control.