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Protein extracts from the heart and liver tissue were obtained by homogenization in lysis buffer (100 mg/mL) as mentioned previously [1 (link), 26 (link)]. The protein concentrations were determined by Lowry protein assay and the samples were electrophoresed in a 12% SDS polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were then transferred to PVDF membranes (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The membranes were blocked using 5% non-fat milk for 1 h. Monoclonal primary antibodies were diluted 1:1000 in antibody binding buffer (TBS) and used for hybridization overnight (4 °C) with the following antibodies ANP, phosphor-Akt, β-Actin, Bax, BNP, Cytochrome C, phosphor-GATA4, PGC1α (sc-20158, sc-7985, sc-47778, sc-526, sc-18818, sc-13560, sc-32823-R, sc-13067, Santa Cruz); phosphor-AMPK, Cle-Caspase-3, phosphor-FOXO3a, Sirt1 (#2535, #9664, #9466, #9475s, Cell Signaling, The Netherlands). Following hybridization with appropriate secondary antibodies the membranes were washed in for 10 min thrice. The blots were detected in chemiluminescent detection using ECL with Fujifilm LAS-3000 (GE Healthcare Life Sciences).

The powdered skeletal muscle tissue (n = 12 animals per time point and genotype, 6 males and 6 females) was resuspended in RIPA lysis buffer with protease inhibitors (Complete, Roche) and the homogenate were centrifuged at 10.000 g for 10 min at 4°C. The supernatants were collected and the protein concentration was quantified by BCA method (Sigma-Aldrich). Forty micrograms of protein was resolved on a 15% SDS-page gel and the proteins were transferred to PVDF membranes (Amersham Biosciences). The membranes were blocked in Tris-buffered saline supplemented with 0.1% Tween 20 and 5% (w/v) powered skim milk overnight at 4°C and then incubated with primary antibodies for one hour at room temperature (RT) against LC3 (1:1000; PD014, MBL), p62/SQSTM1 (1:1000; BML-PW9860, Enzo Life Sciences), Beclin1 (1:500; sc-11427, Santa Cruz), Caspase-3 (1:500; 9662, Cell Signalling), Caspase-9 (1:500; 9508, Cell Signalling), Bax (1:1000; sc-526, Santa Cruz), Bcl2 (1:1000; sc-492, Santa Cruz), PARP-1 (1:500; sc-7150, Santa Cruz) and GAPDH (1:1000; sc-25778, Santa Cruz). After incubation with HRP-conjugated secondary antibodies, bands were visualized by ECL reagents (GE Healthcare Life Science). Inmunoblots were scanned and densitometry was measured with AlphaEase FC software (Bonsai). Results were normalized to the corresponding GAPDH signal.