Western blot analysis.

Mouse hearts and other organs were harvested, snap frozen, and crushed in liquid nitrogen. The tissue was then homogenized in cold lysis buffer (Cell Signaling, Danvers, MA) as previously described (1, 2). Isolated CMs were snap frozen in liquid nitrogen. Cold lysis buffer was added, and the lysate was collected. Protein concentrations were measured by the Bradford method (Bio-Rad, Hercules, CA). SDS-PAGE was performed under reducing conditions on 4–20% gradient gels (Bio-Rad). Proteins were transferred to a PVDF membrane (Bio-Rad). After being blocked with 1% fish gelatin buffer (Sigma-Aldrich), membranes were incubated overnight at 4°C with primary antibodies. Blots were then incubated with donkey anti-rabbit IRDye-680RD or donkey anti-mouse IRDye-800CW secondary antibodies (LI-COR) for 1 h. Signals were visualized on an Odyssey scanner (LI-COR). Primary antibodies against cleaved caspase-3 (Cell Signaling), light chain 3B (LC3B; Cell Signaling), mTOR (Cell Signaling), Akt (Cell Signaling), phospho-Akt (Ser⁴⁷³, Cell Signaling), S6 ribosomal protein (Cell Signaling), phospho-S6 ribosomal protein (Ser^235/236, Cell Signaling), hemagglutinin (Santa Cruz Biotechnology, Dallas, TX), TfR1 (ThermoFisher Scientific, Waltham, MA), ferroportin (Novus Biologicals), and GAPDH (Santa Cruz Biotechnology) were used for immunoblot analysis.