Measuring Autophagic Flux via LC3-II

The LC3‐II flux assay was performed as previously reported with minor modifications (Su et al., 2013 (link)). In brief, mice of the ISO and the CTL groups were subjected to an intraperitoneal injection of 3 μmol/kg BafA1 (Bafilomycin A1, #11038, Cayman Chemical Company) or vehicle CTL (DMSO in saline) 1 h before the ventricular myocardium was sampled for western blot analyses of LC3‐II. The LC3‐II flux is the difference of LC3‐II protein levels between the samples with or without BafA1 treatment of the same group, referring to the net amount of LC3‐II accumulated by BafA1‐induced inhibition of lysosomal function. The LC3‐II flux assays are considered the “gold standard” method for reflecting autophagic activity (Gottlieb et al., 2015 (link)).