Bafilomycin A1 autophagy-flux assay

For in vitro studies, NRCMs were cultured as described above, stimulated with cGMP (50 μM) for 15 minutes, and then with either vehicle or bafilomycin A1 (BFA, 1 μM) (Sigma) for 3 hours. For in vivo studies, WT, SA/SA, and SE/SE mice received two injections of BFA (3 μM/kg, IP) 90 minutes apart. Myocardium was collected 90 minutes after the second injection, and protein extract examined by immunoblot for LC3-II. The relative increase in expression with versus without BFA indexes autophagic flux.