Fig. 5

GLS mediates EV release in mouse astrocytes. a Wild-type and Gls−/− mouse astrocytes were acquired. Protein levels of GLS, including two GLS allozymes, kidney type glutaminase (KGA, 65 kDa) and glutaminase C (GAC, 58 kDa), were analyzed by Western Blot. GFAP was a marker of astrocytes. Actin was used as a loading control. b Wild-type and Gls−/− mouse astrocytes were treated with TNF-α (50 ng/ml) for 24 h in serum-free media. EVs were isolated from the supernatants, and EV protein lysates were prepared. c, d The levels of Alix and Flotillin-2 were determined by Western Blots. Quantification results of Alix and Flotillin-2 were shown as mean ± SD. EVs were isolated from normalized volumes of serum-free supernatants of control and TNF-α treatment group for 24 h. e EVs were isolated and resuspended with PBS, and then the concentration of EVs was analyzed by NTA. *, **, and *** denote p < 0.05, p < 0.01, and p < 0.001 in comparison to control, respectively. ^## and ^### denote p < 0.01 and p < 0.001 in comparison to TNF-α treated groups, respectively. ns denotes no significance