Krüppel-Like Factor 4 Is a Regulator of Proinflammatory Signaling in Fibroblast-Like Synoviocytes through Increased IL-6 Expression

Figure 6

KLF4 and NF-κB-p65 cooperatively induce the IL-6 promoter. KLF4 and NF-κB-p65 cooperatively induce IL-6 promoter activity. RA FLSs were cotransfected with the indicated IL-6 promoter luciferase plasmids, pcDNA3.1-NF-κB-p65 expression plasmid (p65), pcDNA3.1-KLF4 expression plasmid (KLF4), or the pcDNA3.1 empty vector (Neo). IL-6 promoter activity was measured by Dual Luciferase Assay (a). Data were expressed as means ± SEM (). in comparison to the p65 group. KLF4 does not affect either expression or nuclear translocation of NF-κB-p65. RA FLSs were transfected in triplicate with pcDNA3.1-KLF4 expression plasmid (KLF4) or pcDNA3.1 empty vector (Neo) and then incubated with TNF-α (20 ng/mL) for 30 min. Expression of NF-κB-p65 (p65) in the cytoplasm and nucleus was detected by western blot. Cyto: cytoplasmic extracts; NE: nuclear extracts. Histone 3 (H3) and Tubulin served as controls for nuclear and cytoplasmic proteins. KLF4 associates with NF-κB-p65. Nuclear extracts from RA FLSs were immunoprecipitated (IP) with anti-KLF4, anti-NF-κB-p65 (p65), or anti-IgG antibodies, followed by immunoblotting (IB) with the indicated antibodies (c).

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