--- - |2 Abstract Bcl‐2‐associated athanogene3(BAG3) protects the heart and cardiomyocytes from ischaemia/reperfusion (I/R) injury. Although theanti‐apoptosis effect of BAG3 has been demonstratedin multiple cell types, the structural domain of BAG3,which is responsible for its anti‐apoptosis effect, is not well understood. BAG3 protein consists of various characteristic amino acid motifs/regions that permit the interaction of BAG3 with numerous proteins involved in many cellular key pathways.The purpose of this study is to determine whether theproline‐rich (PXXP) domain of BAG3 is necessary for its cellular protection againsthypoxia‐reoxygenation (H/R) stress by binding to its chaperone,heat shock cognate 71 kDa protein (HSC70).Cell apoptosis induced by H/R was evaluated usingpropidium iodide (PI) staining, caspase 3/7 activation and TUNEL stainingin cultured H9C2 cells. The expression levels of BAG3 and HSC70 were manipulated, whereBAG3 or its mutant, which lacked the PXXP domain, wasoverexpressedusing aplasmid and adenovirus vector, and HSC70 expression was silenced usingsiRNA. Co‐immunoprecipitation(co‐IP) followed by western blot was employed to define the complex of BAG3 binding to itschaperones.The PXXP domain of BAG3 was determined to be critical for BAG3‐mediated attenuation of H9C2 cell apoptosis induced by H/R through the binding of PXXP with HSC70.The abolished cellular protection of BAG3 induced bythe knockdown of HSC70 is associated with reduced binding to HSC70. Given that the structural domain PXXP of BAG3 is necessary for the cellular protection of BAG3 from I/R injury, the mechanism revealed in this study indicates that BAG3 may bea therapeutic target in patients undergoing reperfusion after myocardial infarction. This article is protected by copyright. All rights reserved. - 'Clinical and Experimental Pharmacology and Physiology, Volume 0, Issue ja, -Not available-. '