1 d analysis software

The molecular characterization of the WF proteins and protein fractions was performed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), using Laemmli’s method [61 (link)] as described by Alu’datt et al. [62 (link)]. The WF or lyophilized protein fractions (3 mg) were dissolved in 1 mL of a solubilizing buffer, containing 0.5 mL of β-mercaptoethanol, 1 mL of bromophenol blue (1%), 0.6 mL of Tris-HCl (1 M, pH 6.8), 2 mL of 10% SDS (10%), 5 mL of glycerol (50%) and 0.9 mL of distilled water, followed by denaturation in a boiling water bath for 3 min with stirring. Electrophoresis was performed with 4–20% Mini-Protean TGX Precast Gels in a Mini-Protean Tetra Cell (Bio-Rad Laboratories, Hercules, CA, USA), with a migration buffer containing SDS (0.1%), glycine (0.192 M) and Tris-HCl (0.025 M, pH 8.3), and a migration voltage increasing from 60 to 120 V. The gels were stained with a solution of modified Coomassie Brilliant Blue G 250, destained and dried before scanning and analysis (Bio-Rad GS-800 densitometer and Quantity One 1-D Analysis Software, respectively). The molecular weight (MW) of the proteins was determined from a standard curve of the migration distance of MW markers (19–118 kDa) vs. the log of MW. Their corresponding bands were categorized as major or minor bands based on the densitometric intensity (greater or less than 50%, respectively).

Western blot analysis was carried out using different cell extracts and heart samples with different antibodies. Mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg) and hearts were dissected out for extraction of total proteins. Cultured H9C2 was washed three times with PBS and then used for extraction of total proteins. Protein extracts were prepared by lysis in 20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1% DOC, 0.5% NP-40, 10% glycerol, 1 mM glycerophosphate, 1 mM NaF, 2.5 mM Na pyrophosphate, 1 mM Na₃VO₄, and a cocktail of protease inhibitors (Calbiochem) at 4 °C. Protein extracts were mixed with the reducing laemmLi sample buffer, boiled for 12 min, separated by SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. The membranes were cut into two or more horizontal strips from the top to the bottom based on the predicated molecular weights of the target protein(s) and controls, and then blotted individually with appropriate primary antibodies and appropriate secondary antibodies. Images from western blot analysis were captured and quantified using 1-D Analysis Software and Quantity One (Bio-Rad, Hercules, CA, USA). The experiments were repeated at least three times. Full-image scan results of western blots are shown in Supplementary Figs. 16–20.