Figure 2.

ETS2 binds to EBS in the 5′ UTR and induces siah1 transcription and protein expression in the H. pylori-infected GCCs. (a) Promoter and 5′ UTR analysis of human siah1 gene shows that an EBS located between +92 and +95 (represented by a box). We assume that the most upstream exon 1 of the Siah1 cDNA is at position +1²². (b) Western blot results showing the status of ETS2 binding with the siah1 5′ UTR (n=3) in the presence or absence of H. pylori. ETS2 binds to the WT EBS only but not with the EBS-Mut oligo. Western blot of nuclear lysates shows the levels of ETS2 protein expression in the input lanes. HDAC1 is the loading control for nuclear lysates. (c) ChIP assay of ETS2 immunocomplex for siah1 EBS. IgG= immunoglobulin G; M= MW marker; NS= non-specific primer, S= specific primer. (d) Figure shows dual luciferase assay involving WT and ETS2-Mut siah1 5′ UTR-transfected and infected or uninfected MKN45 cells. Data are analyzed by two-way ANOVA with Tukey’s post hoc test (n=3). Error Bars, s.e.m. ***P< 0.001, **P< 0.01, *P< 0.05. (e) Bar graph of dual luciferase assay result showing transcriptional activation of WT siah1 5’ UTR with ectopic ETS2 expression and H. pylori infection. Data are analyzed by two-way ANOVA with Tukey’spost hoc test. Error bars, s.e.m. ***P<0.003; ****P< 0.0001. (f) Transient transfection of ETS2 siRNA followed by western blotting shows Siah1 suppression in the ETS2-suppressed MKN45 cells.