Fig. 2

C/EBPβ binds to delta-secretase promoter and regulates its transcription. a The model of C/EBPβ putative binding sites on human AEP promoter. b, c ChIP assay was performed to detect the binding sites of C/EBPβ on the AEP promoter. The DNA-protein crosslinking ChIP samples from HEK293 cells treated with OGD or not, were immunoprecipitated with anti-C/EBPβ antibody or IgG. After reversing crosslinks, PCR (b) and real-time PCR (c) were performed by using primer pairs at −1540 (site 1), −1480 (site 2), and −330 (site 3) of the human AEP promoter. PCR assay also detected each input sample (b). The positive control PCR experiment was done by using anti-Histone H3 antibody and GAPDH primers (b). In real-time PCR analysis, the anti-C/EBPβ enrichment was represented by the fold of C/EBPβ antibody enriched target DNA comparing to IgG enriched (c, *P < 0.05, Student’s t-test). d HEK293 cells were transfected with pcDNA, C/EBPα, and/or C/EBPβ, as well as human AEP promoter luciferase reporter plasmid (−2029 to +3) or its mutation (Site 2 was mutant). Forty-eight hours later, OGD/reperfusion was performed and then promoter activities were determined by luciferase assay (*P < 0.05, Student’s t-test). e Nuclear proteins were isolated from HEK293 cells received OGD/recovering treatment or not. EMSA assay was recruited to detect the C/EBPβ binding ability increasing on Site 2 of AEP promoter after OGD/reperfusion treatment. f HEK293 cells were transfected with pan-C/EBP decoy DNA or its mutation, and Site2 decoy DNA or its mutation. Forty-eight hours later, cells were treated with OGD/recovering or not. EMSA assay were then performed to detect C/EBPβ binding abilities on Site 2 of LGMN promoter within each sample of nuclear protein. Data are representative of three independent experiments