Journal of Rheumatic Diseases

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Fig. 3. TNF treatment activates NF-κB protein phosphorylation to induce DKK1 mRNA expression in enthesis. Control enthesis cells were stimulated with TNF on days 1 and 3, and analyzed by (A) RT-PCR for DKK1 and GAPDH, and (B) immunofluorescence for DKK1 at day1. (C) Control enthesis cells were stimulated with TNF on days 1 and analyzed by immunoblotting as indicated proteins. (D) Control enthesis cells were co-transfected with three types (WT, S536A mutant, and S536E mutant) of NF-κB, followed by treatment with TNF as indicated doses for 24 hours, and analyzed for luciferase activity (n=4). (E) Control enthesis cells were co-transfected with two types (1 kb and 0.35 kb) of the human DKK1 promoter and beta-gal, followed by treatment with TNF as indicated doses for 24 hours, and analyzed for luciferase activity (n=4). (F) Human primary synovial cells were stimulated with 25 ng/mL TNF for 24 hours, followed by ChIP assays with the phos S536 NF-κB antibody (n=3). (G) Control enthesis cells were stimulated as indicated for a day and analyzed by immunoblotting. Each dot displays an individual value. Values represent means±standard deviations. TNF: tumor necrosis factor, AS: ankylosing spondylitis, ALP: alkaline phosphatase, WT: wild type, ChIP: chromatin immunoprecipitation. *p<0.05, **p<0.01.
J Rheum Dis 2021;28:216~224 https://doi.org/10.4078/jrd.2021.28.4.216
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