Revert total protein staining

Membranes (0.45 µm nitrocellulose, BioRad, USA) were blocked in Blocker™ FL Fluorescent Blocking Buffer (ThermoFischer, Aus) for 1 h at RT. All antibodies (α-syn (1:5000), BD biosciences; β-actin (1:10,000), CST; synaptophysin (1:5000), CST) were diluted in TBS-T. Membranes were washed in TBS-T for 21 min (3 × 7 min) before and after incubation with fluorescent secondary antibodies (Licor, Aus). Proteins were visualized with the Licor Odyssey fc system (Licor, Aus) and analyzed via signal intensities (ImageStudio 5.2, Licor, Aus). Samples normalized to β-actin, except for 4-HNE which was normalized to total protein as determined by Revert Total Protein Staining (Licor, Aus) according to manufacturer’s direction.

Ventricular cardiomyocytes isolated from mouse hearts or hiPSC-CMs were homogenized in ice-cold RIPA buffer using a Potter homogenizer (RW20 digital, IKA). The homogenates were centrifuged at 10,000 × g for 10 min at 4 °C to remove insoluble contents and the protein concentrations determined by bicinchoninic acid assay (Pierce BCA Protein Assay; Thermo Fisher Scientific). Samples were heated for 5 min at 96 °C in 1x Laemmli buffer. Equal amounts of protein samples were loaded and separated on 4–12% gradient SDS-PAGE gels (Bio-Rad) by electrophoresis. Proteins were transferred onto polyvinylidene difluoride membranes (0.45 mm, Immobilon-FL, Merck Millipore). Protein transfer onto membranes was visualized by REVERT total protein staining (LI-COR). Membranes were immunoblotted with primary antibodies overnight at 4 °C. JP2 (NT), JP2 (CT), Calpain-1 and Calpain-2 antibodies were diluted 1:500. JP2 (M), β-Actin and α-Spectrin antibodies were diluted 1:1000. CAPNS1 and RyR2 antibodies were diluted 1:2500. Primary antibodies were then probed with secondary antibodies diluted 1:15,000 overnight at 4 °C. Proteins were visualized using the LI-COR Odyssey CLx imaging station. Protein bands of interest were quantified using the ImageJ software.