Fig. 4

The TRAX/DISC1/GSK3β (TDG) complex is involved in the protective effect of an A_2AR agonist. a, b PC12 cells were treated with an inhibitor of protein kinase A (H89; 10 μM and KT5720; 10 μM) or vehicle for 30 min and then treated with CGS (10 μM) or vehicle for 1 h (a) and 8 h (b). Cells were lysed and subjected to SDS–PAGE, followed by Western blot analysis using the anti-GSK3β Ser9, anti-GSK3β, anti-β-catenin and anti-α-Tubulin antibodies as indicated. The amount of target protein was quantified and normalized to that of α-Tubulin, the loading control. These experiments were repeated three times. c–e In PC12 cells and human MSN neurons, cells were infected with lentivirus expressing GSK3β or a constitutively active GSK3β mutant (GSK3β-S9A) for 3 days. Cells were treated with an A_2AR agonist (CGS21680, CGS; 10 μM) or vehicle for 1 h to activate the A_2AR and then treated with H₂O₂ (100 μM) for 4 h. Cells were lysed and subjected to SDS–PAGE, followed by Western blot analysis using the anti-γH2AX, anti-α-Tubulin and anti-β-Actin antibodies as indicated. The amount of target protein was quantified and normalized to that of α-Tubulin or β-Actin, the loading controls. These experiments were repeated three times (c, d). Apoptosis was assessed by co-staining with Annexin V-Cy5 and PI for 10 min followed by flow cytometric analysis (e). f Protein–protein interactions were monitored by the proximity ligation assay [31] using the corresponding antibodies as described in the Methods section [31]. Each red dot represents the detection of protein–protein interaction. Scale bar, 10 μm. g PC12 cells were treated with an A_2AR agonist (CGS21680, CGS; 10 μM) or vehicle for 1 h. Cells were lysed, subjected to immunoprecipitation with anti-DISC1 and immunoblotted with anti-DISC1, anti-TRAX and anti-GSK3β antibodies. The amount of target protein was quantified and normalized to that of the input. These experiments were repeated three times. h PC12 cells were infected with the lentivirus expressing A_2AR_253–410-Flag (M7) for 3 days. Cells were lysed, subjected to immunoprecipitation with an anti-A_2AR antibody or a control IgG, followed by Western blot analysis using the indicated antibody. The amount of TRAX that interacted with A_2AR was quantified and normalized to that of the input. These experiments were repeated three times. i PC12 cells were infected with lentivirus expressing A_2AR_253–410-Flag for 3 days. Cells were treated with CGS (10 μM) or vehicle for 1 h to activate the A_2AR and then treated with H₂O₂ (100 μM) for 4 h. Cells were lysed and subjected to SDS–PAGE, followed by Western blot analysis using the anti-γH2AX, anti-Flag and anti-α-Tubulin antibodies as indicated. The amount of target protein was quantified and normalized to that of α-Tubulin, the loading controls. These experiments were repeated three times. j PC12 cells were treated with CGS (10 μM) during the indicated time period. Cells were subjected to immunostaining with an anti-TRAX antibody (Green). Scale bar, 10 μm. k PC12-shTRAX cells were transfected with TRAX-V5 and ΔNLS TRAX-V5 plasmids. Cells were treated with CGS (10 μM) or vehicle for 1 h to activate the A_2AR and then treated with H₂O₂ (100 μM) for 4 h. Cells were subjected to immunostaining with anti-γH2AX (Green) and anti-V5 (Red) antibodies. Scale bar, 10 μm. Data are presented as the mean ± SEM from at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control