Anti ldlr

Cell or mouse muscle and brain extracts were prepared in lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, halt proteinase inhibitor cocktail (ThermoFisher Scientific) and halt phosphatase inhibitor cocktail (ThermoFisher Scientific), and subjected to western blot analysis with anti-LC3 (Novus Biologicals, NB100-2220), anti-SQSTM1 (Abnova, H00008878-M01), anti-Aβ42 (Invitrogen; 700254), anti-APP (Biolegend; 803001), HRP-conjugated GFP antibody (Santa Cruz Biotechnology, sc9996), anti-HA (Cell Signaling Technology, C29F4), anti-ATG7 (Sigma Aldrich, A2856), anti-LDLR (Abcam, ab52818), anti-LRP1 (Abcam, ab92544), and anti-ACTB/β-actin-HRP (Santa Cruz Biotechnology, sc47778 HRP) antibodies. The band intensity was analyzed using the ImageJ software.

Before lung excision, the right ventricle was infused with at least 20 ml of sterile 0.9% saline to remove any residual blood in the pulmonary vasculature. Lung tissues were lysed using either nucleoprotein or cytoplasm protein extraction kit (KeyGEN BioTECH, Nanjing, China), respectively. Protein extracts were analyzed on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA). Primary antibodies used in the study were: Anti- phospho-NFκB p65 (Cell Signaling, Boston, MA), anti-Lamin B1 (Bioworld, Louis Park, MN), anti-β-actin (Zhongshan, Beijing, China), anti-ABCA1 (Santa Cruz Biotechnology, CA), anti-TLR2/4, anti-CYP27A1, anti-LXR-α and anti-LDLR (Abcam, Hongkong, China).

Dapagliflozin was purchased from the MedChemExpress Bio-Technology company (HY-10,450). A 10 mM stocking solution was dissolved and stored at -80˚C. RPMI 1640 medium and foetal bovine serum (FBS) were purchased from Gibco, Invitrogen (Carlsbad, CA, USA). Trypsin/EDTA was purchased from HyClone (Logan, UT, USA). WST-8 cell proliferation and cytotoxicity assay kit was purchased from Dojindo (CK04, Kumamoto, Japan). A cholesterol detection assay kit was purchased from Beijing Applygen Technologies (E-1015). The phalloidine staining reagent was purchased from the Sigma-Aldrich company. Nile acid was purchased from Macklin Inc (49,409). The siRNA specific for KLF-5 was designed and provided by the Genomeditech Company (Shanghai). Reagents for real-time PCR were purchased from Takara.
Primary antibodies are detailed as follows: Anti-ABCA1 (Abcam, Cambridge, MA, UK) for WB and IHC. Anti-KLF 5 (Abclonal, China) for WB and IHC. Anti-podocin (Abcam, UK); anti-nephrin (Abcam, UK); anti-LDLR (Abcam, UK); Anti-HMGCR (Abcam, UK); Anti-podocin (Abcam, UK); anti-Bax (Abcam, UK); anti-Bcl-2 (Abcam, UK); anti-caspase 3 (Abcam, UK); and, anti-GAPDH (Proteintech, China). Rats were purchased from the Animal Experimental Centre of Shandong University. All other materials and reagents were endotoxin-free and supplied by the central lab of Shandong Provincial Hospital.

Simvastatin was obtained from Nanjing Duly Biotechnology Co., Ltd. (Nanjing, China). Lunasin was prepared in our laboratory by using recombinant DNA technology as previously described [39 (link)]. Dil-LDL was obtained from Yiyuan Biotechnologies (Guangzhou, China). Lipofectamine 3000 reagent, MEM, opti-MEM, and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti-LDLR, anti-HNF-1α, and anti-PCSK9 antibodies were purchased from Abcam (Cambridge, UK). Anti-β-actin was obtained from Cell Signaling Technology (Danvers, MA, USA). Analysis kits for TC and LDL-C were purchased from Jiancheng Biotechnologies (Nanjing, China). The 4,6-diamidino-2-phenylindole (DAPI) dye was purchased from KeyGEN BioTECH (Nanjing, China). RNAiso plus reagent, the reverse transcription kit, and SYBR^® Premix Ex Taq ™II were obtained from Takara Bio (Shiga, Japan).

Total protein was extracted from peripheral blood mononuclear cells (PBMCs). The proteins were separated with 10% SDS-PAGE and were then transferred to PVDF membranes (Bio-Rad). Immunoblotting was performed by using primary antibodies against LDLR (anti-LDLR; 1:1000, Abcam, Cambridge, MA, USA), anti-LOX-1 (1:1000, Abcam), and anti-GAPDH (1:5000, Santa Cruz Biotechnology, Dallas, TX, USA) and HRP-conjugated secondary antibodies against mouse or rabbit IgG (1:10000, Santa Cruz). Luminescence was detected by using the Fujifilm LAS-3000 image detection system, and image processing and data quantification were performed by using Multi Gauge v.2.02 software (FUJiFILM, Tokyo, Japan). LDLR expression levels were normalized to those of GAPDH (as a loading control), and LOX-1 expression levels were normalized to those of its proform.