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. 2010 Dec 15;152(2):730–740. doi: 10.1210/en.2010-0899

Fig. 5.

Fig. 5.

C/EBPβ is a downstream mediator of NADPH oxidase produced ROS signaling in HESCs. A, Cultured HESCs were untreated or treated for 24 h with 8-br-cAMP in the presence or absence of apocynin (Apo) and/or DPI. Cytosolic/nuclear extracts were prepared and blotted for SRC, STAT5, FOXO1, and C/EBPβ. The expression of lamin B and α-tubulin was assessed to confirm the integrity of the cell fractionation. β-Actin was used as a loading control. B, HESCs were treated for 24 h with 8-br-cAMP in the presence or absence of Apo, and DNA binding of CEBPβ contained in the nuclear extracts was analyzed by EMSA. Nuclear lysates were incubated with excess of [32P]-labeled C/EBP, OCT1 (xs nonspecific) and unlabeled C/EBP (xs specific) oligonucleotides. Both OCT1 and unlabeled C/EBP oligonucleotides served as DNA binding competitors to verify binding specificity. For supershift analysis, nuclear extracts were incubated with an antibody against C/EBPβ before addition of the hot probe. Solid arrowhead indicate position of specific complexes; open arrowhead marks the position of uncomplexed DNA probe. C, Undifferentiated HESCs were transfected with NT siRNA or siRNA targeting p22PHOX and NOX4 and treated 2 d later with 8-br-cAMP. DNA binding of C/EBPβ contained in the nuclear extracts was analyzed by EMSA. To verify DNA binding, binding specificity and for supershift analysis, nuclear lysates were incubated with excess of [32P]-labeled C/EBP, OCT1, and unlabeled C/EBP oligonucleotides. For supershift analysis, nuclear extracts were incubated with an antibody against C/EBPβ before addition of the hot probe. Solid arrowhead indicate position of specific complexes; open arrowhead marks the position of uncomplexed DNA probe. D, Primary cultures were transfected with C/EBP-pGL4, pSG5-C/EBPβ (encoding for full length C/EBPβ), and pCH110 (encoding for β-galactosidase). The cells were left untreated or treated with 8-br-cAMP, Apo, or the combination of 8-br-cAMP and Apo for 24 h. Subsequently firefly luciferase activity was measured. *, P < 0.01; **, P < 0.001.