2.1. Immunoprecipitation of acetylated proteins

All animal protocols were approved by the Institutional Animal Care and Use Committee at the University of Colorado Denver. Male Dahl Salt Sensitive (DSS) rats (Envigo) were fed a normal salt diet (Teklad; 2020×; 0.4% NaCl) or a high salt diet (Teklad; 2020×; 4% NaCl) for 10 weeks and myofibrils were isolated from the left ventricles. Two micrograms of myofibril protein was incubated with antibodies directed against acetylated-lysine epitopes (5.0 μL, PTM Biolabs) overnight at 4 °C. A 100 μL aliquot of protein G agarose (Sigma) was then applied to the sample and allowed to rotate overnight at 4°C. Supernatants were removed and the beads were washed 5 times with TBS-T. The sample was boiled for 5 min in SDS loading buffer and then applied to 10% SDS-PAGE gels, where the sample was run into the gel approximately 3 cm. The gel was then coomassie stained for 10 min and destained overnight. Protein bands were excised from the gel and digested with trypsin, as previously described [15]. Additionally, acetyl-peptide enrichment was utilized following the manufacturers protocol (PTM Biolabs).