Fig. 3

RFX1 regulates IL-17A expression through binding to the IL17A promoter. a The two predicted binding sites of RFX1 in the IL17A promoter. Site 1 is located at ~468–452 bp, and Site 2 is located at ~639–623 bp, both upstream of the TSS of IL17A. b Luciferase reporter vectors with or without binding Site 1 and/or Site 2 are shown to the left. Jurkat cells were transfected with the reporter plasmids and either pcDNA3 empty vector (white bars) or RFX1 expression plasmid (black bars). Cells were lysed 48 h after transfection, and firefly luciferase activity was measured and normalized to Renilla luciferase activity. Each experiment was performed in Jurkat cells in triplicate (n = 3), and values are given as the mean ± s.d. *P < 0.05; **P < 0.01; n.s., not significant, compared between the indicated groups. P-values were determined using two-tailed Student’s t-tests. c EMSA was used to detect the binding of RFX1 protein with binding Site 1 and Site 2 in the IL17A promoter in vitro. The biotin probe specific for Site 1 or Site 2 was bound by nuclear protein extracts of CD4⁺ T cells, which could be blocked by an RFX1 antibody. d ChIP assay was used to analyze the in vivo relevance of RFX1 binding to the IL17A promoter. Gel electrophoresis bands indicated the binding of RFX1 in the promoter region of IL17A. Three independent experiments were repeated in c, d