Immunoblotting

After isolating the myofilament fraction, protein concentration was determined using a BCA assay (Thermo Fisher). Samples were prepared in SDS Tris-Glycine Buffer (Life Technologies) supplemented with Bolt Reducing Buffer (Fisher Scientific), heated for 10 min at 95 °C, and run on 4–12% gradient Tris-glycine gels (Invitrogen). The proteins were transferred onto a nitrocellulose membrane (Thermo Scientific). Following transfer, membranes were incubated with Revert Total Protein Stain (LI-COR Biosciences) for 5 min to assess equal loading and then blocked in a 1:1 ratio Intercept Blocking Buffer (LI-COR Biosciences) to Tris-Buffered Saline (TBS) solution for 1 h at room temperature. The following primary antibodies and dilutions were used and incubated in blocking buffer with 0.1% Tween overnight at 4 °C with gentle rocking: BAG3 (Proteintech, 10599-1AP, 1:5,000), BAG3 (Santa Cruz Biotechnology, sc-136467, 1:500), c-Myc (Cell Signaling Technology, 71D10, 1:1000), HSPB8 (Proteintech, 15287-1AP, 1:3,000), HSP70 (Proteintech, 10995-1AP, 1:3,000), Ubiquitin (Cytoskeleton Inc., AUB01, 1:400), CHIP/STUB1 (Santa Cruz Biotechnology, sc-133083, 1:100), P62 (Proteintech, 18420-1AP, 1:3,000), GAPDH (Cell Signaling Technology, 14C10, 1:4000), Sarcomeric α-actin (Millipore Sigma, A2172, 1:10000). Blots were analyzed using the LI-COR Image Studio software.