Lc3 1 2

Following RIC or control treatment, mouse hearts were excised and cut transversely to separate the atrium and ventricle. Ventricular tissue was then used for western blot experiments. Stored heart tissue was homogenized using lysis buffer (1% NP40, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 2.5 mM Na₄O₇P₂, 1 mM β-glycerolphosphate, 1 mM Na₃VO₄ and a cocktail of protease inhibitors (Roche)). The homogenate was centrifuged at 10,000×g at 4°C for 30 min to obtain cytosolic protein. An equal amount of protein (30 µg protein) from each sample was separated by 10% SDS–polyacrylamide gels and transferred onto nitrocellulose membranes (Bio-Rad). Membranes were incubated with Atg5 (catalog #8540 – Cell Signaling Tech.), phosphor-mTOR (Ser2481) (catalog #2974 – Cell Signaling Tech.), beclin-1 (catalog #3495 – Cell Signaling Tech.), p62 (catalog #5114 – Cell Signaling Tech.), LC3I/II (NB100-2331 – Novus Biologicals), phospho-AMPK (Thr 172) (catalog #2531 – Cell Signaling Tech.), cathepsin L (catalog #AF1515 – R&D Systems) or GAPDH (catalog #G8795 - Sigma-Aldrich Inc.) overnight at 4°C. The membranes were then washed and subsequently incubated with peroxidase-conjugated secondary antibody (Santa Cruz, CA) and detected with the ECL plus Detection Kit (Amersham, Piscataway, NJ). Immunoblots were scanned using an Odyssey LI-COR and quantified using Image Studio (Ver 2.1).

Quadriceps muscle samples (n = 10) from rapamycin- and chloroquine-treated 18–20 month old wild-type and VCP^R155H/+ knock-in mice were harvested and extracted using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific, Rockford, IL). Protein concentrations were determined using the Nanodrop according to the manufacturer’s protocols. Equal amount of proteins were separated on Bis-Tris 4–12% NuPAGE gels according to manufacturer’s protocols. The expression levels of proteins were analyzed by Western blotting using LC3-I/II (Novus Biologicals, Littleton, CO), p62/SQSTM1, mTOR substrates, ubiquitin, optineurin, VCP, and TDP-43 primary antibodies (Abcam, Cambridge, MA). Equal protein loading was confirmed by β actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) staining.