Lastly, to ambiguously demonstrate the interactions between Nrdp1 and USP8 and between USP8 and HIF-1α in OGD-treated neurons, we performed co-immunoprecipitation experiments. Since Nrdp1 targets USP8 for ubiquitylation, we speculated that overexpression of Nrdp1 could enhance protein ubiquitylation and USP8 degradation in PC12 cells. To test this, we pulled down ubiquitylated species from PC12 cell extracts, and then detected the levels of protein ubiquitilytion in the presence of proteasome inhibitor MG132 as well as the protein levels of Nrdp1 and USP8. As shown in Figure 5A, overexpression of Nrdp1 by Ad-Nrdp1 significantly increased the whole levels of protein ubiquitylation in comparison to Ad-green fluorescent protein (GFP) control, and this change was accompanied by decreased USP8 protein levels.