Anti p enos

Total eNOS and p-eNOS protein levels were determined in LV homogenates as previously described [43 (link)], and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Novus Biologicals, Littleton, CO, USA)).
Subsequent to SDS-PAGE, the proteins were transferred to nitrocellulose membranes, and the blots were probed with primary anti-eNOS (1:500, BD Transduction Laboratory, San Jose, CA, USA), anti-p-eNOS (1:1000 Cell Signaling, Danvers, MA, USA), anti-GAPDH (1:10,000, Imgenex), and secondary rabbit anti-mouse immunoglobulin G (IgG) antibody conjugated with horseradish peroxidase (HRP; 1:1000, Santa Cruz Biotechnology, Dallas, TX, USA). All blots were analyzed using the Odyssey system (LI-COR, Lincoln, NE 68504, USA).

HPMEC or monocytes were lysed in PhosphoSafe Extraction Reagent (Novagen, Merck Biosciences, Nottingham, U.K.) and centrifuged for 5 min at 16,000 g at 4°C to collect the supernatant. Protein concentration was determined (BCA Protein Assay Kit, Pierce; ThermoScientific), and thirty micrograms of each sample were electrophoresed on a Criterion XT Bis-Tris Gel 4–12% (Bio-Rad) and transferred to a polyvinylidene difluoride membrane (Millipore, Saint-Quentin en Yvelines, France). The membrane was blocked with 5% w/v skim milk powder in TBST (0.1 M Tris-HCl pH 8,1.5 M NaCl and 1% Tween-20) for 2 h at room temperature, and subsequently incubated with anti-TREM-1 (AbD Serotec), anti-(p)ERK1/2, anti-(p)eNOS, anti-(p)P65 (Nuclear Factor-κB p65), and anti-His (Cell Signaling, USA) antibodies overnight at 4°C. After vigorous washing in TBST, the membrane was incubated with a secondary antibody conjugated to horseradish peroxidase for 1h at room temperature. Immunocomplexes were detected with the SuperSignal West Femto Substrate (Pierce; ThermoScientific). Non-phosphorylated forms or tubulin (Cell Signaling) were used for normalization. Acquisition and quantitative signal density analyses were performed by a LAS-4000 imager (FSVT) and Multi-Gauge software (LifeScience Fujifilm, Tokyo, Japan).

Five‐μm‐thick sections were deparaffinized, rehydrated, and incubated in 1% BSA for 2 h at room temperature. The sections were incubated with anti‐nitrotyrosine (Abcam, Inc., Cambridge, MA, USA) and anti‐p‐eNOS (Ser1177, Cell Signaling Technologies, Inc., Danvers, MA, USA) for overnight at 4°C. Thereafter, the sections were labeled using anti‐mouse IgG‐TRITC and anti‐rabbit FITC (Sigma, St Louis, MO, USA). Finally, the samples were counterstained with DAPI, mounted, and captured using EVOS M5000 Cell Imaging System (Thermo Fisher Scientific, MA, USA).

Proteins were extracted from heart tissue and myocytes. Total protein concentrations of the samples were tested using a BCA protein assay kit. The immunoblots were probed with the following antibodies overnight at 4°C: anti-κ-OR (Santa Cruz, USA), anti-pAMPK, anti-pAkt, and anti-peNOS (Cell Signaling, Beverly, USA). The corresponding secondary antibodies were incubated at room temperature for 1 h subsequently. Band intensities were quantified by densitometry.

EA.hy926 cells were treated with different concentrations (0, 6, 20, and 60 μM) of DPHC. After 24 h, cells were washed and harvested with ice-cold PBS and lysed with a lysis buffer on ice for 1 h. Lysates were centrifuged at 12,000 rpm for 20 min, and the protein concentration in the supernatant was evaluated with a BSA protein assay kit (Bio-Rad, Hercules, CA, USA). Next, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with 10% gel. Proteins were transferred onto a nitrocellulose membrane. Membranes were incubated at 4 °C overnight with the following primary antibodies added separately: anti-β-actin (sc-47778, Santa Cruz Biotechnology, CA, USA; 1:1000), anti-p-AKT (sc-377556, Santa Cruz Biotechnology; 1:1000), anti-p-eNOS (#9571S, Cell Signaling Technology, Danvers, Massachusetts, USA; 1:1000), and anti-p-PI3K (#17366S, Cell Signaling Technology; 1:1000) dissolved in 5% skim milk. Immunoblots were incubated for another 2 h at room temperature with specific secondary antibodies (1:3000). Protein bands were ultimately developed and photographed using the FUSION SOLO Vilber Lourmat system, Paris, France. The Image J 1.50i software (NIH, USA) was used for quantifying the band intensities.

Antibodies include anti-KCa3.1 (Abcam, ab229593, and Alomone Labs, #ALM-051), anti-p-eNOS (Cell Signaling Technology, #9571s), t-eNOS (Cell Signaling Technology, #9572s), anti-TM (Proteintech, 14318-1-AP), anti-NOX2 (Proteintech, 19013-1-AP), anti-SOD1 (Proteintech, 10269-1-AP), anti-CD31 (Proteintech, 66065-1-Ig), GPx1 (Cell Signaling Technology, #3206S), anti-4HNE (Abcam, ab46545), anti-β-actin (Santa Cruz, sc-47778), and anti-GAPDH (Santa Cruz, sc-47724).