Ab172479

Homogenates were prepared from frozen gastrocnemius aliquots using RIPA buffer (Merck Millipore, Temecula, CA, USA). Thereafter, the protein concentrations in the homogenates were determined with a bicinchoninic acid protein assay kit (Takara Bio Inc.) with bovine serum albumin as the standard. From each homogenate, 20 μg of protein was separated on 10-12% SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad laboratories, Inc., Hercules, CA, USA). Primary antibodies against FoxO1 (#2880S; Cell Signaling Technology, Boston, MA, USA) and atrogin-1/Fbx32 (#EPR9148(2), ab168372; Abcam, Cambridge, UK), MuRF1 (#EPR6431(2), ab172479; Abcam) and HRP-linked secondary antibodies against rabbit-IgG (#2729S; Cell Signaling Technology) were used. Blots were detected using the chemiluminescence reagent, ECL Prime Western Blotting Reagent (GE Healthcare, Buckinghamshire, UK), and signal intensities were quantified using a C-DiGit blot scanner (LI-COR bioscience, Lincoln, NE, USA, USA).

The GA muscle was used to quantify the protein expression of muscle markers of synthesis (Akt), regeneration (MyoD and, myogenin), and degradation (LC3B-II, p62, MuRF and myostatin) by Western blot. The muscle samples were homogenized with a lysis buffer and the homogenate's protein concentration was determined by the Bradford assay. Muscle proteins were separated on 14-16% polyacrylamide gel electrophoresis, transferred to a PVDF membrane and, after blocking nonspecific sites, incubated overnight at 4 °C with the specific primary antibodies: Akt (1:1000), phosphorylated Akt (1:1000), MyoD (1:1000; Sigma, SAB4300397), myogenin (1:2000; Sigma, SAB2501587), LC3B-II (1:500; Cell Signaling, 2775), p62 (1:500; Cell Signaling, 5114) MuRF-1 (1:1000; Abcam, AB172479) and myostatin (1:1000; Abcam, AB203076). Then, primary antibodies were detected with a secondary antibody against rabbit, mouse, or goat by the chemiluminescence system. The Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression was used to normalize expressions. Raw data is available in the Additional file 1 attached. The densities of the specific bands were quantified with an imaging densitometer using Image J software.