Figure 5. Binding of C/EBPϵ to the promoter region of the human PHGPx gene.

(A) HL60 cells were incubated with or without 50 ng/ml TNFα for 8 h, and nuclear protein was then extracted. Proteins binding to the DNA probe containing the C/EBP-binding sequence were detected by EMSA. To identify a protein binding to the probe, anti-C/EBPα, C/EBPβ, C/EBPγ, C/EBPδ or C/EBPϵ antibody or the non-radiolabelled probe was mixed and incubated for 1 h at 4 °C with the nuclear protein extract before performing the binding reaction with the radiolabelled probe as a competition experiment. The arrowhead indicates DNA–protein complex. Reproducibility of these results was confirmed in two experiments. (B) HL60 cells incubated for 8 h with or without 50 ng/ml TNFα, fixed with 1% (v/v) formaldehyde, lysed and subjected to immunoprecipitation using the anti-C/EBPα, C/EBPβ, C/EBPγ, C/EBPδ or C/EBPϵ antibody. DNA was released from immunoprecipitates by proteinase K treatment, and the DNA was amplified by PCR using primers corresponding to the promoter region from −247 to −34 of the PHGPx gene. Reproducibility of these results was confirmed in two experiments.