An antibody against β actin

The total protein content of cultured cells was extracted using RIPA buffer containing phenylmethanesulfonylfluoride (PMSF). A BCA protein assay kit (Beyotime, Haimen, China) was used to determine the protein concentration. Proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes. After blocking, the membranes were incubated overnight at 4 °C with diluted (1:300) primary antibodies (polyclonal rabbit anti-polβ; Proteintech). Following extensive washing, membranes were incubated with diluted (1:3000) horseradish peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz). Signals were determined with a chemiluminescence detection kit (Amersham Pharmacia Biotech, Piscataway, NJ). An antibody against β-actin (Santa Cruz Biotechnology) served as an endogenous reference.

Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco-BRL (Carlsbad, CA, USA). Primary rabbit polyclonal antibodies against human phospho-JNK1/2, phospho-ERK1/2, phospho-p38, phospho-p53, JNK1/2, ERK1/2, p38, cytochrome c, PARP, Bax, Bak, and caspase-9 were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA), and an antibody against β-actin was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The antibodies were used at a 1:2,000 dilution. The JNK inhibitor SP600125, was purchased from Calbiochem^® (Merck Millipore, La Jolla, CA, USA). Polyvinylidene difluoride membrane (PVDF) was purchased from Millipore (Billerica, MA, USA). Cellular DNA Fragmentation ELISA kits were purchased from Roche (Basel, Switzerland), the DeadEnd™ Fluorometric terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) system was purchased from Promega Corporation (Madison, WI, USA), and the PrimeScript Reverse Transcription (RT) Enzyme Mix kits and SYBR^® Green polymerase chain reaction (PCR) reagent were purchased from Takara Biotechnology Co., Ltd. (Osaka, Japan). All the reagents used throughout the study were of analytical or cell culture grade purity. Chrysotile asbestos was obtained from Mangya Moutain (Qinghai Province, China).

Protein extracts of HUVEC and HMEC‐1 cells that were exposed to HG or OxLDL alone or in combination were prepared by RIPA lysis buffer (Millipore) containing protease and phosphatase inhibitor. The cell lysates were first resolved on SDS‐PAGE gels and transferred to polyvinyldene difluoride membranes by electroblotting. Membranes were incubated with 1:500 diluted monoclonal antibodies against eNOS, phosphorylated eNOS (p‐eNOS) (Santa Cruz), ECE, ETA, ETB, Nrf2, pNrf2 and CD31 (Abcam) in PBS for one hour at room temperature. The membranes were then washed thoroughly in Tris‐buffered saline contained 0.1% (v/v) Tween 20 before a second incubation for one hour at room temperature with a 1:1000 diluted horseradish peroxidase‐conjugated secondary antibody (Santa Cruz). An antibody against β‐actin (Santa Cruz) was used to normalize protein loading. The resultant bands were quantified by densitometry.

Primary antibodies against p21, CDK2, Cyclin D1, CDK6, Mcl-1, Bcl-xL, Bcl-2, Bax, Poly (ADP-Ribose) Polymerase (PARP), phospho-AKT (Ser473), AKT, phospho-mTOR (Ser2448), mTOR, phospho-70S6K (Thr421/Ser424), 70S6K and LC3B were purchased from Cell Signaling Technology (Beverly, MA, United States). An antibody against β-actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). The secondary antibodies, including goat anti-rabbit IgG-horseradish peroxidase (HRP) and goat anti-mouse IgG-HRP, were obtained from Bio-Rad (United States). Dimethyl sulfoxide (DMSO), 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 3-methyladenine (3-MA), chloroquine (CQ) LY294002, bafilomycin A1 (Baf-A1) and rapamycin (Rapa) were purchased from Sigma Company (St. Louis, MO, United States). IATL with the purity≥98% as determined by high performance liquid chromatography was bought from Chengdu Must Bio-Technology Co. Ltd. (Chengdu, China). The chemical structure of IATL is shown in Figure 1A.

Total protein from cultured cells was extracted using RIPA buffer containing phenylmethanesulfonyl fluoride. Protein concentrations were determined with BCA protein assay kit (Beyotime, Haimen, China). Extracted proteins were subjected to SDS‐PAGE gels and then transferred to polyvinylidene difluoride membranes. After blocking, the membranes were incubated overnight at 4°C with diluted (1:1000) primary antibody (polyclonal rabbit anti‐polβ). Following extensive washing, the membranes were incubated with diluted (1:3000) horseradish‐peroxidase‐conjugated goat anti‐rabbit IgG (Santa Cruz Biotechnology). Signals were detected using a chemiluminescence detection kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). An antibody against β‐actin (Santa Cruz Biotechnology) served as an endogenous reference.