Fusion sl system

Expression of C301S/C303S-ALDH2 in the absence and presence of cycloheximide was determined by immunoblotting. Cells were harvested and homogenized by sonication (3x5s) in 10 mM Tris buffer, pH 7.4, containing 125 mM KCl, 5 mM EGTA, 2 mM MgCl₂, and Complete Protease Inhibitor Cocktail. Protein concentrations were determined with the Pierce BCA Protein Assay Kit using bovine serum albumin as standard. Denatured samples (10 μg) were separated by SDS-PAGE on 10% gels and transferred electrophoretically to nitrocellulose membranes. After blocking with 5% nonfat dry milk in phosphate-buffered saline containing 0.1% (v/v) Tween-20 for 1 hour, membranes were incubated overnight at 4°C with a primary polyclonal antibody to human ALDH2 (1:20 000; kindly provided by Dr. Henry Weiner) or to β-actin (1:200 000; Sigma). Thereafter, membranes were washed three times and incubated for 1 hour with a horseradish peroxidase-conjugated anti-rabbit (ALDH2) or anti-mouse (β-actin) IgG secondary antibody (1:5000). Immunoreactive bands were visualized by chemiluminescence using ECL detection reagent (Biozym Scientific, Hessisch Oldendorf, Germany) and quantified densitometrically using the Fusion SL system (Peqlab, Erlangen, Germany).

Transfected EA.hy926 cells (siControl, siMCU/EMRE or siMICU1) were incubated with vehicle (water) or 100 μM ATP at 37 °C for 3 or 5 min. Cells were then harvested and homogenized by sonication (3×5 s) in ice-cold RIPA lysis buffer (Sigma, Vienna, Austria) containing 2 mM EDTA, protease and phosphatase inhibitors (CompleteTM, PhosSTOPTM, Roche, Vienna, Austria). Protein concentration was determined with the PierceTM BCA Protein Assay Kit using bovine serum albumin as standard (Fisher Scientific Austria GmbH, Vienna, Austria). Denatured samples (30 μg) were separated by SDS-PAGE on 10% gels and transferred electrophoretically to nitrocellulose membranes. After blocking with 5% non- fat dry milk in Tris-buffered saline containing 0.1% (v/v) TWEEN-20 for 1 h, membranes were incubated overnight at 4 °C with a primary antibody against eNOS (1:2000; BD Transduction Laboratories), Ser¹¹⁷⁷ phospho-eNOS (1:1000; BD Transduction Laboratories) or β-actin (1:200,000; Sigma). Thereafter, membranes were washed 3 times and incubated for 1 h with a horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (1:5000). Immunoreactive bands were visualized by chemiluminescence using ECL detection reagent (Biozym, Germany) and quantified densitometrically using the Fusion SL system (Peqlab, Erlangen, Germany).