Figure 1

(a) Detection of BAG3 protein in supernatants from cultured cardiomyocytes. Human (HCMa) and rat (H9c2) cardiomyocytes, at 80% confluence, were incubated with or without 10% FBS for 16 h at 37 °C in a 5% CO2 atmosphere. Supernatants were dialyzed in a buffer containing 50 mmol/l NaCl and 0.05% IGEPAL, lyophilized, resuspended in 1 ml of RIPA buffer (50 mmol/l Tris HCl pH 7.6, 150 mmol/l sodium chloride, 2 mmol/l sodium orthovanadate, 4 mmol/l EDTA, 10 mmol/l sodium pyrophosphate, 1% NP-40, 0.1% sodium deoxycholate) and analyzed with anti-BAG3 or anti-GAPDH antibodies by western blotting. (b) Mass analysis of BAG3 detected in the sera from two patients affected by CHF. The sera were analyzed with the anti-BAG3 polyclonal antibody TOS-2 in western blotting. (c) ELISA test for detection of anti-BAG3 antibodies in chronic HF patients. Serum samples from 52 CHF patients (EF<45%) and from 84 healthy donors were analyzed for the presence of anti-BAG3 antibodies by ELISA. Results are plotted as arbitrary units (A.U.). Bars in the dot plot depict the median value obtained in the analyzed groups