Fig. 1: BAG3 is important for maintaining cellular protein quality control.

a Reduced expression of BAG3 disrupts cellular proteostasis and causes accumulation of aggregated proteins. Cardiomyocytes were transduced with Ad-GFP, Ad-BAG3, and Ad-siBAG3 for 48 h. Expression of BAG3 was determined in the total cellular fraction. b–d Soluble and insoluble fractions were prepared from the transfected cardiomyocytes and expression of p62, HSP70, and ubiquitin was determined by western blotting. Expression was normalized with Tubulin. e Representative images show that knockdown of BAG3 causes accumulation of ubiquitin-positive cellular aggregates in cardiomyocytes. Cardiomyocytes were transduced with the respective adenovirus and cells were fixed with 4% PFA. Fixed cells were subsequently stained with ubiquitin antibody (red) and nuclei were stained with DAPI. f–h Inhibition of proteasomal function upregulates BAG3 expression in the cardiomyocytes. Cardiomyocytes were treated with MG132 (20 μM) for 6 h and expression of BAG3 was evaluated by western blot. Expression of p62, HSP70, and ubiquitin was assessed by western blot in cardiomyocytes treated with MG132 for 6 h. g–h Knockdown of BAG3 increases accumulation of ubiquitinated proteins in the insoluble fraction. NRVCs were transduced with adenovirus for 48 h and treated with MG132 before protein isolation. Western blot was done with ubiquitin antibody in the soluble and insoluble fractions. i Representative microscopy images show that inhibition of proteasomal function causes upregulation of BAG3 protein in cardiomyocytes. Cardiomyocytes were treated with MG132 for 6 h and cells were fixed with 4% PFA. Fixed cells were then stained with BAG3 antibody (green) and nuclei were stained with DAPI (blue). j Fixed cells were stained with p62 antibody (red) and nuclei were stained with DAPI