Protein Extraction and Western Blot Analysis

Protein from cell cultures were extracted by freeze-and-thaw cycles. Muscles were cut into 40 slices, 20 μm thick and lysed with 100 μl of lysis buffer (50 mM Tris pH7.5, 150 mM NaCl, 10 mM MgCl2, 0.5 mM DTT, 1 mM EDTA, 10% glycerol, 2% SDS, 1% Triton from Sigma-Aldrich®, 1:50 Complete Phosphatase inhibitor from Roche®). The total protein concentration was determined using BCA kit (Abcam®, Prodotti Gianni, Milano, Italy). Cell lysates were subjected to SDS-polyacrylamide gel electrophoresis and then western blotting. Primary antibodies were anti-p-AKT (Thr308, cat# 13038), anti-p-mTOR (Ser2448, cat# 2971), anti-p-S6 (Ser 235/236, cat# 4858), monoclonal anti-GAPDH (cat# 5174), anti-LC3 (cat# 4108), anti-P62 (cat# 8025, all from Cell Signaling®; dilution 1:1000), anti-Lys63 (cat# 05-1308), and anti-Lys48 (cat# 05-1307, both from Millipore®; dilution 1:500) (25 (link)). Immunoreactive bands were detected using proper HRP-secondary reagent and Clarity Western ECL Substrate (Bio-Rad).