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Figure 3


Fig. 3. Snail2 activates its own promoter as a transcriptional activator. (A) A dose-dependent activation of D0.1-Luciferase reporter gene by Snail2 in NIH3T3 cells. Different amounts of Snail2 expression vector were co-transfected with the Snail2 promoter-Luciferase. (B) Snail2 protein binds to the E-Box2 sequence. Isotope-labeled probe containing the E-Box2 was incubated with Snail2 protein, and subjected to EMSA. The shifted band is diminished by a preincubation of the Snail2 protein with cold wild-type competitor, but persists by a preincubation with E-Box2-mutated competitor. (C) E-box2 is required for the Snail2 promoter activation by Snail2. When E-box2 in the D0.1 reporter is mutated (Em2), activation level of the promoter by Snail2 (+) is significantly decreased, compared with the wild type. (D) Snail2 acts as a transcriptional activator on the Snail2 promoter. An expression vector of VP16 activation domain and Snail2 zinc-finger motifs (VP) strongly activates the D0.1 promoter, while an expression vector of Engrailed2 repression domain and Snail2 zinc-finger fusion (En) has no significant effect on the Snail2 promoter activity. Wt, wild type Snail2. (E) Semi-quantitative RT-PCR analysis of endogenous Snail2 expression in neural plate explants, transfected with VP16-Snail2 and cultured without BMP4. Expression level is normalized with the value of amplified GAPDH. Result obtained from explants cultured for 18 hours with BMP4 are also indicated. (F) Transfection of En-Snail2 and VP16-Snail2 into ectoderm inhibits the expression of endogenous Snail2 protein in cultured embryos (arrowheads). GFP-fluorescence indicates the transfected area. Compare with the un-transfected, left neural folds. As the anti-Snail2 antibody recognizes the N-terminal sequence, it detects only endogenous Snail2 protein. (G) Transfection of VP16-Snail2 inhibits the induction of endogenous Snail2 expression in neural plate explants treated with BMP4.





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