Fig. 6

EVs isolated from the cortex after CSD contain HMGB1. A Western blots of cortical EV lysates isolated with a commercial kit validated that the vesicles contained HMGB1 in addition to the EV marker protein TSG101 and neuronal marker CD171. Neuronal EVs subsequently separated with CD171-conjugated beads contained higher amounts of HMGB1. Western blots of neuronal EVs confirmed that they do not contain astrocyte- (ALDH1L1, S100B) and microglia-specific (Iba-1) markers, unlike the brain lysate used as a positive control. B The brain harvesting procedure itself caused considerable vesicular HMGB1 release, as seen in Western blots of the sham groups. However, HMGB1 in EVs was significantly higher in the CSD group than in the sham surgery group (Mann‒Whitney U test, p = 0.038). In the graph, each dot represents an individual mouse cortex subjected to optogenetically or pinprick-induced CSD (purple and pink dots, respectively). Whole membrane images of the Western blots are given in Additional file 2: Fig. S2.