Horseradish-peroxidase-conjugated anti-IgG antibodies were used as the secondary antibody to detect the above-mentioned protein bands by enhanced
chemiluminescence WESTSAVE-Up (Abfrontier). RNA extraction was achieved using 1 mL TRIzol reagent. The RNA pellets were washed in 70% ethanol, dried completely, and dissolved in diethylpyrocarbonate to inhibit RNase. Total RNA was quantified using a ND-100 spectrometer (NanoDrop Technologies, Wilmington, DE, USA). Polymerase chain reaction ON-01910 cost (PCR) was performed using the synthesized cDNA as a template and using specific primers for COX-2 or β-actin as a loading control. The primer sequence for human COX-2 was 5′-GACAGTCCACCAACTTACAAT-3′ (forward) and 5′-CATCTCTCCATCAATTATCTGAT-3′ (reverse). The amplified products were resolved by 1% agarose gel electrophoresis, stained with ethidium bromide,
and photographed under ultraviolet light. HUVECs were cultured in a glass culture chamber slide (Falcon Plastics, London Ontario, Canada) and processed for immunofluorescence analysis. Immunofluorescence was performed as described previously . The amount of prostaglandin (PG)E2 in the culture medium was measured using the PGE2 EIA kit according to the manufacturer’s protocol (Cayman Chemical Company, Ann Arbor, MI, USA). Samples as well as standards were applied to a 96-well plate, precoated with goat anti-mouse IgG, and incubated with PGE2 acetylcholinesterase http://www.selleckchem.com/products/17-AAG(Geldanamycin).html tracer and PGE2 antiserum. All the wells were emptied, rinsed five times, and incubated with Ellman’s reagent for 60 min in the dark with gentle rocking to produce 5-thio-2-nitrobenzoic acid, which has a strong absorbance at 405 nm; the plate was read at 405 nm in an enzyme-linked immunosorbent assay reader Nintedanib (BIBF 1120) (EL
800; Bio-Tek, Winooski, VT, USA). We calculated the results using the standard curve, which were expressed as picograms per milliliter. Intracellular ROS in acrolein-stimulated HUVECs is analyzed using a fluorescent dye, 2′,7′-dichlorofluorescein diacetate (DCF/DA). In the presence of oxidants, DCFH was converted to the highly fluorescent DCF. After 18 h incubation with 25 μM acrolein in the presence or absence of KRG, cells were stained with 10 μM DCF/DA, and fluorescence was analyzed by a FACS Vantage flow cytometer (Becton Dickinson, San Jose, CA, USA) and fluorescence microscopy (Eclipse 50i; Nikon, Japan) . To clarify whether KRG-mediated inhibition of acrolein-induced COX-2 expression plays a significant role in cytoprotection against oxidative stress, acrolein-stimulated cells were pretreated with KRG (1 mg/mL) or untreated, and cell death was measured by in situ terminal transferase dUTP nick end labeling (TUNEL) assay.