Sodium orthovanadate

Catalase activity was measured by a colorimetric assay using Amplex Red Catalase Assay Kit (Molecular Probes) according the instructions provided by the manufacturer. Briefly, hearts were homogenized in lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% SDS), supplemented with 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin A, 1 μg/ml leupeptin, 1 μg/ml aprotinin (Sigma-Aldrich), 10 mM sodium fluorate (AnalaR Normapur; VWR, Leuven, Belgium), and 1 mM sodium orthovanadate (Alfa Aesar, Ward Hill, MA). Supernatants were transferred to microtubes and protein concentrations were determined using the DC protein assay kit (Bio-Rad Laboratories, Hercules, CA). For the assay, 15 μg of total protein were used.

Cells were quantified using a Countess automated cell counter and were seeded into 6 cm plates at 5 × 10⁵ cells/well. Cells were incubated for 6 h in culture media and then resuspended in starvation culture media using DMEM high glucose (Wisent, St-Bruno, Quebec) with 0.1% FBS, 100 U/mL penicillin/streptomycin, and 2.50 μg/mL amphotericin-B for 18 h. Incubation was followed by administration of single agent carboplatin (15 μM), rapamycin (10 nM) or everolimus (10 nM), or a combination treatment for 24 h. Cells were preincubated for 15 min with 2 mM sodium orthovanadate (Alfa Aesar, Haverhill, Massachusetts) as a pre-treatment to preserve phosphorylated proteins. Protein was extracted in complete lysis buffer (Cell Signaling Technology, Danvers, Massachusetts) containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton-X 100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 μg/mL leupeptin, 1 mM PMSF, 2 μg/mL aprotinin, and 1% phosphatase inhibitor cocktail II. Plates were incubated on ice for 5 min followed by cell scraping. Collected protein was incubated for 20 min on ice prior to collection of supernatants. Protein concentration was quantified by the Bradford protein assay (Bio-Rad, Hercules, California) using a bovine serum albumin (BSA) standard curve.

VSMCs were harvested and homogenized in HEPES buffer (130 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM d-glucose and 20 mM HEPES, pH 7.4), supplemented with PMSF 1 mM, pepstatin A 1 µg/ml, leupeptin 1 µg/ml, aprotinin 1 µg/ml (Sigma–Aldrich), sodium fluorate 10 mM (AnalaR Normapur; VWR International), and sodium orthovanadate 1 mM (Alfa Aesar). A total of 200 µl of VSMCs lysate containing 400 µg of protein was mixed with 2 µl of primary antibody (1:100) and the reaction mixture was rotated in a Stuart SB3 Rotator (Camlab Ltd) at a gentle speed overnight at 4°C. The following day, 15 µl of Protein A and 15 µl of Protein G (Santa Cruz Biotechnology) were added to the reaction mixture followed by rotation at 4°C for 4 h. The immunoprecipitation (IP) complex was then washed twice in lysis buffer followed by centrifugation at 14000 rpm for 30 s. The pellet was collected and prepared for immunoblotting.