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Endogenous products of APP metabolism negatively affect HIPK2 DNA-binding activity.

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posted on 21.02.2013 by Cristina Lanni, Lavinia Nardinocchi, Rosa Puca, Serena Stanga, Daniela Uberti, Maurizio Memo, Stefano Govoni, Gabriella D'Orazi, Marco Racchi

(a) ChIP experiments were performed with anti-HIPK2 antibody on HEK-293 and HEK-APP cells that were also treated with β-secretase inhibitor at 1 µmol/L for 48 h and on HEK-293 treated with conditioned medium from HEK-APP cells for 48 h in the absence or presence of β-secretase inhibitor; PCR analyses were performed on the immunoprecipitated DNA samples using specific primers for the human HIF-1α promoter as shown in Figure 1. (b) ChIP experiments were performed with anti-HIPK2 antibody on HEK-293 and HEK-APP cells that were also treated with β-secretase inhibitor at 1 µmol/L for 4 8h and on HEK-293 treated with conditioned medium from HEK-APP cells for 48 h in the absence or presence of β-secretase inhibitor; PCR analyses were performed on the immunoprecipitated DNA samples using specific primers for the MT2A promoter. (c) HEK-293 and HEK-APP cells were transfected with MT2A-luc and HIF-1α-luc reporter construct and luciferase activity was measured 36 h after transfection. Results normalized to β-galactosidase activity are presented as fold of induction of luciferase activity ±S.D. At least three independent experiments performed in duplicate. * p<0.01 (Student t-test). (d) MT2A and HIF-1α mRNA expression was determined in HEK-APP compared to HEK-293 cells by reverse-transcriptase (RT)-PCR. GAPDH was used as loading control.

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