2.3. Western blot analysis

Myofibril-enriched protein (10 μg) was subjected to standard SDS-PAGE using Criterion TGX Stain-Free 8–16% gradient precast gels (Bio-Rad, Hercules, CA) at 180V for 45 minutes, or Mini-PROTEAN TGX Stain-Free 4–20% gradient precast gels (Bio-Rad, Hercules, CA) at 150V for 1 hour for PTMs, 5% SDS-PAGE at 150V for 1 hour for high molecular weight proteins, and 15% SDS-PAGE at 150 V for 80 minutes for low molecular weight proteins. Protein was transferred to an Immobilon^® -P^SQ membrane (Merck Millipore, Burlington, MA). Membranes were blocked using 5% (w/v) nonfat dry milk in Tris-buffered saline–0.1% Tween 20 (TBS-T) or Odyssey® Blocking Buffer (PBS) (LI-COR Biotechnology, Lincoln, NE) for 1 hour at room temperature. Membranes were then immunoblotted with primary antibodies directed against succinyllysine (PTM-419, PTM Biolabs, Chicago, IL), SIRT5 (15122–1-AP, Proteintech Group, Rosemont, IL), aspartate aminotransferase ({"type":"entrez-nucleotide","attrs":{"text":"Ab189863","term_id":"51987981","term_text":"AB189863"}}Ab189863, Abcam, Cambridge, MA), trifunctional enzyme subunit alpha (Ab54477), trifunctional enzyme subunit beta (ARP48133_P050, Aviva Systems Biology, San Diego, CA), periostin (Ab14041), isocitrate dehydrogenase 2 (Ab94359), acetyl CoA acetyltransferase 1 (PA5–34676, Thermo Fisher, Rockford, IL), creatine kinase-MM ({"type":"entrez-nucleotide","attrs":{"text":"Ab174672","term_id":"90086362","term_text":"AB174672"}}Ab174672), ATP synthase F1 subunit beta (Ab14730), succinate dehydrogenase complex flavoprotein subunit A (Ab14715), dihydrolipoamide succinyltransferase (E2) component of the 2-oxoglutarate dehydrogenase complex ({"type":"entrez-nucleotide","attrs":{"text":"Ab177934","term_id":"66863933","term_text":"AB177934"}}Ab177934), succinate CoA ligase subunit alpha (Ab97867), acetyllysine (9441, Cell Signaling, Boston, MA), trimethyllysine (PTM-601, PTM Biolabs), mono- and dimethyllysine (PTM-602, PTM Biolabs), glutaryllysine (PTM-1151, PTM Biolabs), malonyllysine (PTM-901, PTM Biolabs), propionyllysine (PTM-201, PTM Biolabs), and crotonyllysine (PTM-502, PTM Biolabs). Following 3 washes with TBS-T, or Phosphate-buffered saline (PBS)-Tween 20 (0 .1% v/v; PBS-T), a horseradish peroxidase conjugated secondary antibody was applied and membranes were developed using Clarity Western ECL Substrate from Bio-Rad. Chemiluminescence was visualized using a Chemidoc^® MP (Bio-Rad). Succinyllysine and all other PTM blots were also imaged using the Odyssey imaging system with IRDye® 800CW Secondary Antibodies (#926–32213, LI-COR Biotechnology, Lincoln, NE). Protein band intensities were quantified using Image Lab (Bio-Rad, version 5.0) and normalized to the total protein per lane using stain-free gel imaging on a Chemidoc^® MP (Bio-Rad) to ensure equal protein loading.