Bradford protein determination assay

Cells were lysed in RIPA buffer (Boston Bio Products) supplemented with protease and phosphatase inhibitors. Proteins were quantified using the Bradford protein determination assay (Bio-Rad), and 20–30 μg of protein were loaded and resolved on 10% NuPAGE Bis-Tris gels (Life Technologies) then transferred to nitrocellulose membranes (Bio-Rad) before incubation with the indicated antibodies. Proteins were detected using SuperSignal West Pico Chemiluminescent Substrate (Pierce).

Cells were washed 3 times with PBS and lysed in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100 and 0.1% SDS; pH 8.0) with protease and phosphatase inhibitors (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Cell lysates were centrifuged (10,000 × g at 4°C for 10 min). The protein concentration was measured using a Bradford protein determination assay (Bio-Rad Laboratories) and 20 µg protein was loaded per the lane. Proteins were separated on 10% SDS-PAGE gels and blotted onto nitrocellulose membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were blocked in 3% non-fat dry milk for 1 h at room temperature and probed with primary antibodies for FOXO3, pAkt, Akt, p27, LaminB1, GAPDH, cleaved caspase3, Bim (EL), Bax, Bcl2, and β-actin. All antibodies were diluted at 1:1,000 and incubated for 1 h at room temperature. Membranes were then probed with HRP-tagged goat anti-mouse or anti-rabbit IgG antibodies, diluted at 1:15,000 and 1:5,000 respectively, for 1 h at room temperature. Chemiluminescence was detected using ECL.

Ventricles, n = 5⿿7/treatment/sex/diet, were homogenized in RIPA buffer (1% NP-40, 50 mM Tris (pH 7.4), 0.5% deoxycholate, 159 mM NaCl, 0.1% SDS, 10 mM sodium metabisulfite, 1 mM sodium vanadate, proteinase inhibitor cocktail, PhosSTOP (Roche, Indianapolis, IN), and 1 mM PMSF). Protein was measured using the Bradford Protein Determination Assay (BioRad, Hercules, CA) as per the manufacturer⿿s instructions.
Protein expression was measured using standard immunoblotting methods. Primary antibodies to the cardiac calcium homeostasis proteins sodium calcium exchanger-1 (NCX1, Abcam Inc, Cambridge, MA ab6495); sarcoplasmic calcium ATPase 2a (SERCA2a, Santa Cruz Biotechnology, Santa Cruz, CA, sc-8095); cardiac calsequestrin 2 (CASQ2, Abcam Inc, Cambridge, MA, ab626662); phospholamban (PLB, Thermo Scientific, Nepean, ONT, MA3-922); and phospho-serine 16-specific PLB (pS¹⁶-PLB, Millipore, Temecula, CA, 07-052 1) were obtained. Secondary antibodies complexed to horseradish peroxidase and chemiluminescent detection kits were obtained from Pierce Chemical Co., (Rockford, IL). Several exposures from each membrane were collected onto X-ray film. After immunoblotting, membranes were washed, permanently stained with Coomassie Brilliant Blue, destained and scanned. The bands on the X-ray film and stained membrane were scanned and quantitated using Image J software.