Horseradish peroxidase hrp conjugated anti mouse

The soleus fibers were homogenized using a lysis buffer and centrifuged at 13,000 rpm for 20 minutes. Protein concentration was measured via colorimetric protein assay kit (Bio-Rad, Hercules, CA, USA). Proteins (30 µg) were separated on sodium dodecyl sulfate-polyacrylamide gels and transferred onto a nitrocellulose membrane on ice. The membranes were incubated using 5% skim milk with Tris-buffered saline which is containing 0.1% Tween-20 (TBS-T) and then, incubated overnight at 4°C with the following primary antibodies: GAPDH, Bcl-2, Bax, cytochrome c (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Akt, p-AKT, mTOR, p-mTOR, p70S6k, p-p70S6k, 4EBP1, p-4E-BP1, FOXO3, MuRF1, and Atrogin-1 (Cell Signaling Technology, Danvers, MS, USA). The membranes were incubated for 1 hour with secondary antibodies: horseradish peroxidase (HRP)-conjugated anti-mouse (Santa Cruz Biotechnology) or goat anti-rabbit IgG-heavy and light chain antibody HRP conjugated. After washing 3 times in TBS-T, an enhanced chemiluminescence detection kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to detect the band. Protein bands was expressed using a ChemiDoc (Bio-Rad). All protein levels were calculated with GAPDH as a ratio.