Figure 4.

Ang2 induces astrocyte apoptosis via GSK-3β/β-catenin pathway under high glucose (HG; 25 mM glucose). Although astrocytes were incubated for 48 h under HG, Ang2 (300 ng/ml) was treated for the indicated time period. (a) Western blot analysis for phospho-GSK-3β (Ser9) and phosphor-β-catenin (Thr41/Ser45) were performed on lysates obtained from astrocytes treated with Ang2 for 15, 30, and 60 min under HG (48 h). (b) Western blot analysis for β-catenin was performed on lysates obtained from astrocytes treated with Ang2 for 24 and 48 h under total 48 h of HG. (c) After 48 h of HG incubation, SB216763 (GSK-3β inhibitor, 10 μM) was pretreated for 1 h. Then, Ang2 was treated for 30 min. Western blot analysis was performed for phospho-GSK-3β (Ser9) and phosphor-β-catenin (Thr41/Ser45). (d and e) Astrocytes were incubated under HG with either Ang2 or SB 216764 for 48 h. (d) Western blot analysis for β-catenin was performed. (e) Apoptotic cell counts were assessed by FACS analysis. The bar graph represents mean±S.E.M. of three independent experiments. *P<0.05 by Student's t-test. Data represent three independent experiments. β-Tubulin was used as a loading control