Fig. 3 | Molecular Psychiatry

Fig. 3

From: GSK3β negatively regulates TRAX, a scaffold protein implicated in mental disorders, for NHEJ-mediated DNA repair in neurons

Fig. 3The alternative text for this image may have been generated using AI.

TRAX and ATM contribute to the DNA-PK-mediated NHEJ pathway evoked by A2AR activation. a PC12 cells were treated with an A2AR agonist (CGS21680, CGS; 10 μM) or vehicle for 1 h. Following the treatment, cells were treated with H2O2 (100 μM) for the indicated time periods. Cells were lysed and analyzed by immunoblotting with anti-DNA-PKcs T2609, anti-DNA-PKcs and anti-α-Tubulin antibodies. The amount of target protein was quantified and normalized to that of α-Tubulin, the loading control. These experiments were repeated three times. b PC12-pSuper and PC12-shTRAX cells were treated with CGS (10 μM) for 1 h and treated with H2O2 (100 μM) for 1 h. Cells were lysed and analyzed by immunoblotting with anti-DNA-PKcs T2609, anti-DNA-PKcs, anti-TRAX and anti-α-Tubulin antibodies. The amount of target protein was quantified and normalized to that of α-Tubulin, the loading control. These experiments were repeated three times. c PC12 cells were treated with an inhibitor of ATM (KU55933; 10 μM) or vehicle for 30 min and then treated with CGS (10 μM) or vehicle for 1 h to activate the A2AR, followed by the addition of H2O2 (100 μM) for 1 h. Cells were lysed and subjected to SDS–PAGE, followed by Western blot analysis using the anti-DNA-PKcs T2609, anti-DNA-PKcs 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. d Schematic illustration of the NHEJ assay in vivo. A single DSB was generated in this plasmid substrate pEGFP-C3 with NheI to cut between the promoter and the GFP gene. Linearized plasmids and the circular pDsRed-monomer-C1 as the internal control for transfection efficiency were cotransfected into the PC12-pSuper cells and PC12-shTRAX cells. The region of the forward primer binding site is in the promoter and the reverse primer binding site is in the GFP gene. After transfection for 2 days, cells were collected for qPCR. Data are shown as the mean ± SEM from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control

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