Pierce ripa buffer

To determine the respective protein myosin heavy chain (MHC) isoform abundance, muscle samples were incubated with 250 μl Pierce™ RIPA buffer (Thermo Fisher Scientific, MA, USA), mixed with cOmplete™ Mini EDTA-free Protease Inhibitor Cocktail (Roche, Basel, Switzerland), homogenized and incubated for 10 min on ice in Pierce™ RIPA buffer to a final concentration of 5 μg/μl muscle protein. After centrifugation (4°C, 14000 rpm, 20 min), the supernatant was used for SDS-PAGE according to the manufacturer’s instructions (Mini-Protean TGX Gels 4–20%, BIO-RAD, Berkeley, USA). Afterwards, silver staining was performed with the Silver Stain kit (BIO-RAD, #161–0443, Berkeley, USA) according to the manufacturer’s instructions. The relative protein abundance was analyzed with Image J 1.51 (ImageJ, NIH, Maryland, USA).

EVs were lysed in an RIPA buffer (Thermo Scientific, Milan, Italy Pierce RIPA buffer) containing protease inhibitor (Roche, Milan, Italy Protease inhibitor cocktail tablets) and phosphatases inhibitor (Sigma Aldrich Milan, Italy, Phosphatase inhibitor cocktail 3) for 2 h in ice and the protein concentration was determined using the Bradford assay (Bio-Rad, Milan, Italy Protein Assay Dye Reagent Concentrate). Then, 16 μg of protein was separated using 10% SDS-PAGE and blotted onto a nitrocellulose membrane (GE Healthcare, Milan Italy Amersham Protran 0.45 µm NC ). The membranes were stained with a Ponceau S solution (Sigma, P7170-1L) and then washed with Tris-buffered saline containing 0.1% Tween-20 (T-TBS). After blocking with 5% no-fat dry milk in T-TBS, the membranes were incubated at 4 °C overnight with the following primary antibodies: anti-COL1A2 (Abcam, Cambridge, UK ab96723 1:1000), anti-Cytokeratin 2e (KRT2 Abcam, ab170106 1:800), anti-Alix (Santacruz Biotechnology, Texas, USA, 1A12 1:250), anti-Calnexin (Abcam ab10286 1:1000), and anti-TSG101 (Abcam ab83 1:500). Detection was performed using peroxidase-conjugated secondary antibodies using the ultra-enhanced chemiluminescence system (Thermo Scientific, Super Signal West Femto Maximum Sensitivity Substrate #34095).