Western Blot Protein Analysis Protocol

Equal values of protein (150 μg/line) were resolved in SDS-PAGE and relocated to nitrocellulose membranes. Subsequently, the membrane was blocked for 2 h in a saline solution [10 mM Tris (pH 7.5), 150 mM NaCl, 0.05% Tween 20] at room temperature with 5% non-fat milk. Membrane washing (three washes, 10 min each) occurred in saline solution. Then, the membrane was incubated membrane with diluted primary antibody (Akt, mTOR, p70S6k, GSK3, and MAFbx) from Santa Cruz Biotechnology (Santa Cruz, CA, United States) in either 5% w/v BSA, 1X TBS, 0.1% Tween^® 20 at 4°C with gentle shaking, overnight. After a new membrane washing process, the membrane was incubated with an anti-IgG antibody (1:10,000 dilution) attached to horseradish peroxidase (saline solution with 1% non-fat milk) for 1 h. Posteriorly, the incubation of the membrane was performed using a substrate for peroxidase and a chemiluminescence enhancer (ECL Western Blotting System Kit, GE Health Care, Little Chalfont, Buckinghamshire, United Kingdom) for 1 min and submitted to X-ray film. After the development of the films, the intensity of the band was measured by optical densitometry (Hatanaka et al., 2013 (link)).