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. 2011 Aug 3;6(8):e22621. doi: 10.1371/journal.pone.0022621

Figure 3. Identification of a BMP-4-response element in the 5′-flanking region of PV.1A.

Figure 3

(A) Serially deleted promoter constructs were injected (20 pg/embryo) with or without BMP-4 (0.5 ng/embryo) into 2-cell stage embryos. Animal caps were dissected from injected embryos at stage 8.5 and incubated until stage 11 in 0.5X MBS for measurement of luciferase activity. A putative BRE was detected between −180 and −162 bp from the transcription start site based on reporter gene expression. The underlined sequence (CAGA) is a consensus binding-site for Smad proteins. Luciferase activity was measured as described in Materials and Methods. (B) 3BRE is a luciferase fusion construct with a triple BRE repeat. The 3BRE construct (20 pg/embryo) was co-injected with DN-BR or BMP-4 (each 0.5 ng/embryo) into 2-cell stage embryos. Animal caps were dissected at stage 8.5 and incubated until stage 11 in 0.5X MBS for measurement of luciferase activity as described in Materials and Methods. (C) The -180 WT and -180 MT constructs (20 pg/embryo) were injected with or without BMP-4 or Smad1 (each 0.5 ng/embryo) into 2-cell stage embryos. -180 MT indicates a construct with mutated BRE. Luciferase activity was measured as described in Materials and Methods. The sequences underlined indicate alterations in the original sequences. Experiments were repeated three times using independent sample sets. Data are shown as mean ± SD.