Fig. 3

Dual-luciferase reporter assays detected Sp1 binding sites on tgfbr2 promotor. A The sequence logo of the Sp1 binding motif. B Schematic of the 3 predicted Sp1 binding sites on tgfbr2 promotor and their binding sequences accordingly. The binding sites were located at -1135 to -1125 (site 1), -105 to -95 (site 2) and + 19 to + 28 (site 3) of the tgfbr2 promotor. The tgfbr2 luciferase activities tested by applying a series of truncations (C) as well as site-targeted mutations (D) on the tgfbr2 promoter, along with pcDNA3.1-Sp1 and pRL-TK plasmids. The specific constructs used in the truncation assays (C) included pGL3-basic vector, pGL3-tgfbr2-promo-WT (containing promotor region from − 1405 to + 98), pGL3-tgfbr2-promo-truncation1 (from − 1085 to + 98), pGL3-tgfbr2-promo-truncation2 (from − 746 to + 98), pGL3-tgfbr2-promo-truncation3 (from − 402 to + 98), pGL3-tgfbr2-promo-truncation4 (from − 41 to + 98) and pGL3-tgfbr2-promo-truncation4&mut3 (from − 41 to + 98 which lacking site 3). The specific constructs used in the site-mutation assays (D) included pGL3-basic vector, pGL3-tgfbr2-promo-WT (containing all 3 sites), pGL3-tgfbr2-promo-mut1 (lack of site 1), pGL3-tgfbr2-promo-mut2 (lack of site 2), or pGL3-tgfbr2-promo-mut3 (lack of site 3). The luciferase activities were determined and presented as the ratio of firefly luciferase activity and renilla luciferase activity. The assays were performed with 3 replicates, and data were presented as mean ± SEM. **p < 0.01. ns, no significance. E Schematic of the evidenced Sp1 binding site on tgfbr2 promotor and ChIP-qPCR verification of Sp1 binding with the tgfbr2 promotor at the site around + 19 in hBMECs upon infection. The ChIP procedure was performed with an anti-Sp1 antibody, and rabbit IgG was employed as the negative control. The qPCR was assayed with 3 replicates, and data were presented as mean ± SEM. **p < 0.01