Figure 6
Figure 6. ChIP analysis of effects of p65RelA on C/EBP-ϵ binding to lactoferrin (LF) and defensin (HNP) promoters. (A) Chromatin immunoprecipitations were performed from MiaPaca2 cells treated with control lentiviral vector (lanes 1-4, 9-10) or MiaPaca2 cells with siRNA-induced knock down of p65RelA (lanes 5-8). Five days after transduction, cells were transfected with expression plasmids directing expression of 75D, 75A, or 75T variants of C/EBP-ϵ or an empty vector control. At 36 hours after transfection, chromatin immunoprecipitations were performed, using α–/EBP-ϵ or α-p65RelA. The precipitated chromatin was analyzed using primers specific for C/EBP sites in the LF promoter, HNP promoter, or IkBα promoter. Input chromatin (1:20 dilution) is presented in lane 9. In lane 10, input chromatin was derived from immunoprecipitation with a control antibody (α-p65RelA in case of LF and HNP detection, α–C/EBP-ϵ in case of IkBα detection). (B) In vivo effects of C/EBP-ϵ mutations at codon 75 on DNA binding within the LF and HNP promoters. Chromatin immunoprecipitations were performed on RelA expressing HEK293 cells, transfected with expression plasmids for the wild-type C/EBP-ϵ (75T, lane 5), phosphomimetic 75D mutant (lane 3), or 75A mutant of C/EBP-ϵ. Empty vector–transfected control cells were used for ChIP in lane 4. In negative control experiments presented in lane 1, input chromatin was derived from immunoprecipitation of C/EBP-ϵ–transfected HEK293 cells with a control antibody (α-p65RelA in case of LF and HNP detection, α–C/EBP-ϵ in case of IkBα detection). (C) Chromatin immunoprecipitations (ChIPs) and detection of lactoferrin (LF) promoter were performed in mouse 32Dcl3 cells that were transduced with retroviral vector expressing C/EBP-ϵ phosphomimetic D75 mutant (lanes 2-5) or an empty vector control (lane 1). Five days after transduction, cells were treated with control lentiviral vector (lanes 2, 4, 5) or with RelA-specific siRNA vector. ChIPs were performed 3 days after siRNA transduction, with α–C/EBP-ϵ (lanes 1-3) or control α-p65RelA (lane 4).

ChIP analysis of effects of p65RelA on C/EBP-ϵ binding to lactoferrin (LF) and defensin (HNP) promoters. (A) Chromatin immunoprecipitations were performed from MiaPaca2 cells treated with control lentiviral vector (lanes 1-4, 9-10) or MiaPaca2 cells with siRNA-induced knock down of p65RelA (lanes 5-8). Five days after transduction, cells were transfected with expression plasmids directing expression of 75D, 75A, or 75T variants of C/EBP-ϵ or an empty vector control. At 36 hours after transfection, chromatin immunoprecipitations were performed, using α–/EBP-ϵ or α-p65RelA. The precipitated chromatin was analyzed using primers specific for C/EBP sites in the LF promoter, HNP promoter, or IkBα promoter. Input chromatin (1:20 dilution) is presented in lane 9. In lane 10, input chromatin was derived from immunoprecipitation with a control antibody (α-p65RelA in case of LF and HNP detection, α–C/EBP-ϵ in case of IkBα detection). (B) In vivo effects of C/EBP-ϵ mutations at codon 75 on DNA binding within the LF and HNP promoters. Chromatin immunoprecipitations were performed on RelA expressing HEK293 cells, transfected with expression plasmids for the wild-type C/EBP-ϵ (75T, lane 5), phosphomimetic 75D mutant (lane 3), or 75A mutant of C/EBP-ϵ. Empty vector–transfected control cells were used for ChIP in lane 4. In negative control experiments presented in lane 1, input chromatin was derived from immunoprecipitation of C/EBP-ϵ–transfected HEK293 cells with a control antibody (α-p65RelA in case of LF and HNP detection, α–C/EBP-ϵ in case of IkBα detection). (C) Chromatin immunoprecipitations (ChIPs) and detection of lactoferrin (LF) promoter were performed in mouse 32Dcl3 cells that were transduced with retroviral vector expressing C/EBP-ϵ phosphomimetic D75 mutant (lanes 2-5) or an empty vector control (lane 1). Five days after transduction, cells were treated with control lentiviral vector (lanes 2, 4, 5) or with RelA-specific siRNA vector. ChIPs were performed 3 days after siRNA transduction, with α–C/EBP-ϵ (lanes 1-3) or control α-p65RelA (lane 4).

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