Anti lox 1

Cell Counting Kit-8 (CCK-8), Bicinchoninic acid (BCA) Protein Assay Kits, Annexin V-FITC Apoptosis Detection Kits, and Cell Cycle Analysis Kits were all purchased from the Beyotime Institute of Biotechnology (Jiangsu, China). Protein Extraction Kits were obtained from KEYGEN Biotech. Co., Ltd. (Nanjing, China). Diaminobenzidine (DAB) substrate kits were purchased from Zhongshan Golden Bridge Biotechnology (Beijing, China). Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) kits were provided by Roche Diagnostics (Germany). 4′,6′-Diamidino-2-phenylindole (DAPI), tris (hydroxymethyl) aminomethane (Tris), and sodium dodecyl sulphate (SDS) were purchased from Sigma (St. Louis, MO, U.S.A.). Anti-LOX1, anti-caspase-9, anti-p65, anti-p-IKKβ, anti-IKKβ, anti-p-IkBα, anti-IkBα, anti-GAPDH, anti-β-tubulin, and anti-Lamin B primary antibodies, and horseradish peroxidase-conjugated antibody were obtained from Abcam (Cambridge, U.K.). Plasmids harboring the wild-type caspase-9 response element (WT-luciferase-caspase-9) and the corresponding mutant (MUT-luciferase-caspase-9) were purchased from Vipotion Biotechnology (Guangzhou, China). Pierce Agarose Chip Kits were obtained from Thermo Fisher Scientific (Waltham, MA, U.S.A.).

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.

Protein expression of gp91 phox, Cu/Zn-SOD, and LOX-1 in HUVECs was determined by western blot analysis. Briefly, HUVECs were homogenized and centrifuged to extract proteins. Protein concentrations were determined by BCA Protein Assay Kit (P0011, Beyotime Institute of Biotechnology, Nanjing, China) with a microplate reader (SH-1000, Corona Electric Co., Ltd, Japan) at OD 562 nm. Every 30 μg aliquot of protein was separated by a SDS-PAGE gel and transferred onto a nitrocellulose membrane. The transferred membrane was incubated overnight with specific polyclonal antibodies: anti-gp91 phox (1:500, Abcam, USA), anti-Cu/Zn-SOD (1:500, Novus Biologicals, USA), anti-LOX-1 (1:500, Abcam) and anti-β-actin (1:1000, Abcam). After washing three times, the blots were incubated with a corresponding secondary antibody and the probed protein was visualized by luminolchemiluminescenceBeyo ECL Plus (P0018, Beyotime Institute of Biotechnology, Nanjing, China) and finally detected by autoradiography exposure. The membrane was then re-blotted with β-actin antibody as a loading reference. Densitometry of the probed protein bands was analyzed for the aliquotsand normalized to corresponding β-actin, which was expressed as fold increases compared to the normal control group.