Western Blotting

Extracted ALS and healthy vesicles were resuspended in lysis buffer (8 M urea; 2% SDS; 10 μL/mL protease inhibitor cocktail), and protein was extracted from muscle biopsies using RIPA buffer. Extracted proteins were loaded into NuPage polyacrylamide 4–12% BisTris gels for electrophoresis under reducing conditions (Calnexin, Flotillin, ALIX, FUS, SOD1, TDP43, RPL5, and skeletal alpha‐actin) and non‐reducing conditions (CD63 and CD81). Transfer on polyvinylidene difluoride (PVDF) membrane was performed using the iBlot® Dry Blotting System (Life Technologies™) and upon transfer polyacrylamide gels were stained with Blue Coomassie Gel Code Blue Stain Reagent (LifeTechnologies™) to visualize proteins. Immunoblotting was carried out using the iBind™ Flex Western System and primary antibodies (refer to Table S2) with respective secondary antibodies (Goat anti‐Mouse HRP, Donkey anti‐Goat HRP, Goat anti‐Rabbit HRP). The signal was detected using the Amersham ECL™ Prime Western blotting Detection Reagent and the UVP ChemiDoc‐It2 Imager.