Slow myosin

Hematoxylin and eosin (H & E) staining was performed on paraffin-embedded sections of the diaphragm and quadriceps. Immunohistochemistry of paraffin-embedded diaphragm sections was performed using antibodies against BIS [1 (link)], slow myosin, fast myosin (Sigma-Aldrich, St. Louis, MO, USA), cleaved PARP1 (Abcam, Cambridge, UK), HSF1 (Enzo Life Sciences, Farmingdale, NY, USA), and YAP1 (Santa Cruz Biotechnology, Dallas, TX, USA). Cell nuclei were counterstained with DAPI (4′,6-diamidino-2′phenylindole, 1:2000; Roche, Mannheim, Germany) for 10 min in immunofluorescence immunohistochemistry. Bright-field images were captured using Pannoramic MIDI (3DHISTECH Ltd., Budapest, Hungary) and analyzed using CaseViewer 2.4 software (3DHISTECH Ltd.). Fluorescence images were viewed under a confocal microscope (LSM 900 with Airyscan; Carl Zeiss Co. Ltd., Oberkochen, Germany) equipped with four lasers (Diode 405, Argon 488, HeNe 543, and HENe 633) under constant viewing conditions. The CSA, minimum Feret diameter, and percentage of fibers with centralized nuclei were measured from multiple areas using Image J software (Fiji; National Institutes of Health, Bethesda, MD, USA).

Protein extraction from both Wt and MLC/SOD1^G93A transgenic tibialis anterior muscles was performed in Sodium Chloride, 1 mM Phenylmethylsulfonyl fluoride, 1 μg/ml Aprotinin, 1 μg/ml Leupeptin, 1 μg/ml Pepstatin, 1 mM Sodium orthovanadate, 1 mM Sodium fluoride. Equal amounts of protein from each muscle lysate were separated in SDS polyacrylamide gel and transferred onto a nitrocellulose membrane. Filters were saturated with 5% milk and then blotted with antibodies against Slow myosin (1:3000) (Sigma Aldrich, United States), Glut4 (1:250) (Cell Signaling Technology, United States), PDH-E1α (pSer300) (1:400) (Calbiochem, United States and Canada), Pyruvate Dehydrogenase complex (PDH; 1:600) (Cell Signaling, United States), Phospho-GSk3β (pSer9) and total GSk-3β (1:1000) (Cell Signaling, United States), ATGL (1:100) (Cell Signaling, United States), pACC(1:600) (Cell Signaling, United States), ACC(1:250) (Cell Signaling, United States), Plin2 (2μg/ml) (Life Span Biosciences, United States), and α-tubulin (1:2000) (Sigma Aldrich, United States).
Then, filter was incubated with secondary antibodies Goat anti-mouse IgG HRP-conjugated (1:7000) (Bethyl, Montgomery, TX, United States) or Goat anti-rabbit IgG HRP-conjugated (1:7000) (Bethyl, Montgomery, TX, United States) in 1% milk for 1 h. All the antibodies were chosen as validated for western blot by manufactures.