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Background: Ubiquitination supports cell homeostasis and cell survival particularly under stress conditions, e.g. in the presence of reactive oxygen species (ROS). We previously found that focal cerebral ischemia leads to reperfusion-dependent ubiquitin aggregation. The mechanisms driving post-ischemic ubiquitination and whether it contributes to cell survival remain elusive. The proteasome, responsible for degradation of ubiquitinated proteins, is suppressed after ischemia, but it is unclear whether this contributes to ubiquitin accumulation. Here we investigate the relationship between post-ischemic ubiquitination, ROS and proteasome inhibition, and the impact of ubiquitination on post-ischemic cell fate.Methods: Organotypic hippocampal slices (HS) prepared from neonatal male C57BL6 mice underwent oxygen-glucose deprivation (OGD; 95%N2/5%CO2 for 60 min followed by recovery) or H2O2 treatment. Ubiquitination and ROS were assessed by Western blotting or dihydroethidium (DHE), respectively, and proteasome activity either by cleavable proteasome substrates or by an AAV-transfected GFPu reporter in live cells. Cell death was monitored with propidium iodide.Results: OGD increased ubiquitination at 60 min recovery (1.5±0.1-fold of normoxia (Nx), P<0.05, n=3-7), an effect not observed without O2 during recovery, or in presence of ROS scavengers, suggesting involvement of ROS. Accordingly, H2O2-mediated ROS production also induced ubiquitination (1.8±0.2-fold of untreated, P<0.05, n=3-7). OGD suppressed proteasome activity by 49±3% (P<0.05, n=3) and augmented GFPu levels during recovery (1.13±0.03-fold of Nx, P<0.05, n=12), suggesting that post-ischemic proteasome inhibition contributes to ubiquitin accumulation. Finally, treatment with the E1 enzyme inhibitor PYR41 reduced OGD-induced ubiquitination by 51±5% and enhanced cell death (+23% at 24 hours, n=12-18), implying a protective effect of ubiquitination.Conclusions: We identify ROS and proteasome suppression as key players in regulating post-ischemic ubiquitination. Furthermore we establish for the first time that post-ischemic ubiquitination supports cell survival, a finding that may have implications for the development of novel stroke therapies.