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. 2014 Mar 14;289(18):12876–12885. doi: 10.1074/jbc.M113.535831

FIGURE 7.

FIGURE 7.

The DNA binding activity of KLF6 is essential to promote p65 binding to the promoters. A, KLF6-WT and its ZNFs but not its TAD could bind to the promoters of IκBα and CXCL2. HEK293 cells (3 × 106) were transfected with indicated plasmids (5 μg each). Twenty hours after transfection, cells were treated with IL-1β (10 ng/ml) for 30 min. ChIP assays were performed with the indicated antibodies. The binding of Flag-tagged KLF6 or its truncation mutants with indicated promoters were determined by qPCR analysis. V: Vector, W: wild-type of KLF6, N: KLF6(aa1–200), and C: KLF6(aa190–283). B, neither the N terminus nor C terminus of KLF6 potentiates IL-1β-induced NF-κB activation. HEK293 cells (1 × 105) were transfected with the NF-κB luciferase reporter (20 ng) and Flag-KLF6 or its truncation mutants (20 ng each). Twenty hours after transfection, cells were untreated or treated with IL-1β (10 ng/ml) for 10 h before reporter assays were performed. C, wild-type KLF6 but not the KLF6(C265Y) potentiates IL-1β-induced NF-κB activation. HEK293 cells (1 × 105) were transfected with an NF-κB luciferase reporter (0.02 μg) and the indicated plasmids (0.02 μg each). Twenty hours after transfection, cells were untreated or treated with IL-1β (10 ng/ml) for 10 h before reporter assays were performed. The lysates were analyzed by immunoblots with the indicated antibodies. D, wild-type KLF6 but not KLF6(C265Y) mutant potentiates IL-1β-induced transcription of MCP1, CXCL2, and IL8 genes. HEK293 cells (4 × 105) stably transfected with control vector, Flag-KLF6 or Flag-KLF6(C265Y)were untreated or treated with IL-1β (10 ng/ml) for the indicated time points before qPCR analysis was performed. E, KLF6(C265Y) interacts with p65. HEK293 cells (2 × 106) were transfected with HA-tagged p65 (1 μg) and Flag-tagged KLF6 or its C265Y mutant (5 μg each) for 20 h before coimmunoprecipitation and immunoblot analysis was performed with the indicated antibodies. F, wild-type KLF6 but not KLF6(C265Y) mutant could bind to promoters IκBα and IL8 genes after IL-1β stimulation. HEK293 cells (4 × 105) stably transfected with control vector, Flag-KLF6, or Flag-KLF6(C265Y) were stimulated with IL-1β (10 ng/ml) for 30 min. ChIP assays were performed with the indicated antibodies and binding of KLF6 or KLF6(C265Y) to the cognate sites was determined by qPCR. G, overexpression of wild-type KLF6 but not KLF6(C265Y) potentiates IL-1β-induced DNA binding of p65 to promoters of MCP1, CXCL2, and IL8 genes. The experiments were performed as in F except that the p65 antibody was used.