Fig. 7: Psmb8 promotes Drp1 degradation.

A Lysates from siRNA-control- or siRNA-Psmb8-infected NRCMs following H/R 24 h were IP with anti-Drp1 antibody and then analyzed with immunoblottong with anti-ubiquitin (Ub) or anti-Drp1 antibody (left). Input for each protein (right). Quantification of ubiquitinated Drp1 level (right, n = 3). B Lysates from NRCMs infected with Ad-GFP or Ad-Psmb8 and then following H/R 24 h were IP and then analyzed as described in (A). C WT and Psmb8-KO mice were subjected to sham or I/R surgery for 24 h. Heart lysates were immunoprecipitated with an anti-Drp1 antibody. Immunoblotting analysis of ubiquitin-conjugated Drp1 (left). Quantification of the relative ubiquitinated Drp1 protein level (middle, n = 3), and input of Psmb8 and Drp1 proteins (right). D rAAV9-Psmb8 or rAAV9-GFP-injected mice were subjected to sham or I/R surgery for 24 h. Heart lysates were immunoprecipitated and immunoblotting analysis of ubiquitin-conjugated Drp1 (left) as well as input (left) were performed as in (C). E NRCMs were first infected with adenovirus expression siRNA-control or siRNA-Psmb8 for 24 and then stimulated with CHX (10 μM) for another 6, 12 or 24 h. The protein levels of Drp1 and Psmb8 for each time point were determined by immunoblotting analysis with anti-Drp1 or anti-Psmb8 antibody (left) and quantification of the ubiquitinated Drp1 level (right; n = 3). F NRCMs were infected with Ad-GFP or Ad-Psmb8 and treated and analyzed as described in (E). All values are expressed as the mean ± SEM, and n indicates the number of independent experiments or samples per group.