Fig 2: The TRAX/DISC1/GSK3ß (TDG) complex is involved in the protective effect of an A2AR 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-GSK3ß Ser9, anti-GSK3ß, anti-ß-catenin and anti-a-Tubulin antibodies as indicated. The amount of target protein was quantified and normalized to that of a-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 GSK3ß or a constitutively active GSK3ß mutant (GSK3ß-S9A) for 3 days. Cells were treated with an A2AR agonist (CGS21680, CGS; 10 µM) or vehicle for 1 h to activate the A2AR 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-?H2AX, anti-a-Tubulin and anti-ß-Actin antibodies as indicated. The amount of target protein was quantified and normalized to that of a-Tubulin or ß-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 A2AR 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-GSK3ß 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 A2AR253–410-Flag (M7) for 3 days. Cells were lysed, subjected to immunoprecipitation with an anti-A2AR antibody or a control IgG, followed by Western blot analysis using the indicated antibody. The amount of TRAX that interacted with A2AR was quantified and normalized to that of the input. These experiments were repeated three times. i PC12 cells were infected with lentivirus expressing A2AR253–410-Flag for 3 days. Cells were treated with CGS (10 µM) or vehicle for 1 h to activate the A2AR 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-?H2AX, anti-Flag and anti-a-Tubulin antibodies as indicated. The amount of target protein was quantified and normalized to that of a-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 ?NLS TRAX-V5 plasmids. Cells were treated with CGS (10 µM) or vehicle for 1 h to activate the A2AR and then treated with H2O2 (100 µM) for 4 h. Cells were subjected to immunostaining with anti-?H2AX (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