Anti lys48

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).

Protein lysates were collected from regional human astrocytes in radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich) supplemented with a protease and phosphatase-3 inhibitor cocktail (Sigma-Aldrich), then 20 μg of protein was resolved on a 4-12% Tris gel and transferred to a polyvinylidene difluoride (PVDF) membrane using the Trans-Blot Turbo system (Bio-Rad) according to standard protocols. Membranes were incubated overnight at 4 °C in Tris-buffered saline (TBS), 0.1% Tween® 20 (TBST) plus 5% powdered milk and anti-LMP2 (Abcam; ab184172) or anti-Lys48 (Millipore; 05-1307) and anti-β-actin (ThermoFisher Scientific; MA5-15739) antibodies, washed with TBST 3 times, and then incubated with Alexa Fluor 488, 647, or HRP-conjugated secondary antibodies (ThermoFisher Scientific) for 1 h at room temperature. Membranes were washed with TBST 3 times and imaged using the ChemiDoc MP imaging system (Bio-Rad).