2000mm system

The phosphorylated protein extraction was performed using the FOCUS PhosphoRich Kit (G-Bioscience, USA) according to the instructions. Western blotting was performed as previously described. Briefly, equal amounts of protein lysate were separated on 12% SDS gel and then electrotransferred to the PVDF membrane. The membrane was incubated with the different primary antibodies: anti-AKT (Cell Signaling Technology, CST, 1 : 1000), anti-phospho-Akt (Ser473, CST, 1 : 1000), and anti-PAI-2 (Abcam, 1 : 1000 dilution) over night at 4°C. Horseradish peroxidase signals were detected by enhanced chemiluminescence substrate (ECL Plus Western Blotting Detection System, GE Healthcare, Uppsala, Sweden) and captured with a CCD system (image station 2000MM, Kodak, Rochester, NY, USA). The quantitative analysis of these images was performed using Molecular Imaging Software Version 4.0, provided by Kodak 2000MM System [19 (link)]. The optical density was normalized against that of β-actin.

Gastrocnemius muscles were homogenized in RIPA buffer plus Phosphatase Inhibitor and Complete Mini Protease Inhibitor (1 mg protein per 20 mL RIPA) and cell lysates were then centrifuged at 12,000 g for 10 min and the supernatants were collected. The protein concentrations were measured and 100 mg of protein was treated with SDS/DTT loading Buffer A prior to gel electrophoresis as described previously [34 (link)], using the following antibodies myostatin, Atg3, Atg7, Atg12, Beclin-1, LC3-I/II, p-PI3K, PI3K, p-Akt, Akt, p-FoxO3a, FoxO3a, p-FoxO1, MAFbx, MuRF1, PAMA4, Rpt5, Rpn11, MHC, and GAPDH. Images were captured and documented with a CCD system (image station 2000MM, Kodak, USA). The quantitative analysis of these images was performed using the Molecular Imaging Software Version 4.0 provided by Kodak 2000MM System.

Aliquots of heart tissue (50 mg) were homogenized in liquid nitrogen and dissolved in lysis buffer. Protein concentrations were determined by BCA protein quantitative assay. The protein lysates were loaded onto 10% SDS-polyacrylamide gel for separation, electrotransferred to PVDF membranes, and blocked in 5% nonfat milk in Tris-buffered saline, Membranes were incubated overnight using primary antibodies, (caspase-3 (Cell Signaling Technology Inc., no. 9662, USA) diluted to 1 : 1000, p-53 (Bioworld Technology Inc., BS3736, Louis Park, USA) diluted to 1 : 500, fas (Assay Designs, ADI-AAP-221D, USA) diluted to 1 : 500, Bcl-2 (Cell Signaling Technology, no. 2876, USA) diluted to 1 : 1000, and Bax (Cell Signaling Technology, no. 2772, USA) diluted to 1 : 1000) at 4°C. This step was followed by secondary antibodies, which were conjugated using horseradish peroxidase. We performed enhanced chemiluminescence (Merck-Millipore, Germany) detection. The images were captured and documented using a CCD system (image station 2000MM, Kodak, Rochester, NY, USA). Quantitative analysis of these images was performed using Molecular Imaging Software Version 4.0, which was provided by Kodak 2000MM System. The optical density was normalized against actin [10 (link)].