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Fig. 1. (A) Cos-7 cells were transfected with increasing amounts of an expression vector containing the Gal4 DNA-binding domain (DBD) fused to full-length Dach1 (Gal4-WT), along with the 4R-UAS-Luc reporter plasmid, which contains four Gal4-DBD-binding motifs. Transcription was repressed by Dal4-WT in a dose-dependent manner (12.5- to 23-fold repression). (B) Using the same reporter, a low amount of Gal4-Dach1 was expressed in Cos-7 cells, resulting in weak repression. However, co-expression of mouse Sin3a strongly enhanced the repressor activity. (C) Co-expression of Gal4 DBD-Smad1 and BMP4 activated the 4R-UAS-Luc reporter. Expression of Dach1 weakened this BMP4-mediated activation. Addition of mouse Sin3a further repressed this activation. When Dach1 expression was removed, mouse Sin3a-mediated repression was no longer observed. (D) An immunoprecipitation assay was carried out using HA-tagged Dach1 and Flag-tagged mouse Sin3a expressed in Cos-7 cells. An irrelevant IgG did not precipitate either Dach1 or Sin3a (IP IgG). When the anti-HA antibody was used, both Dach1 and Sin3a were detected (IP Dach1=HA) by the anti-Flag antibody (IB Flag=mSin3a) and the anti-HA antibody (IB HA), respectively. Input, 5% of the extract used in this immunoprecipitation assay. (E) Flag-tagged Smad1 and HA-tagged Dach1 were co-expressed in Cos-7 cells, and cell lysates were immunoprecipitated with the anti-HA antibody or an irrelevant IgG (as a negative control). In the precipitant of the irrelevant IgG, neither Smad1 nor Dach1 was detected (IP IgG). By contrast, both Smad1 and Dach1 were present in the precipitant when the anti-HA antibody was used for IP (IP Dach1=HA). Input, 5% of the extract used in this immunoprecipitation assay. (F) Addition of Dach1 repressed the BMP-4/Smad1/Smad4-mediated transcription of the Xvent2-Luc reporter. Co-expression of VP=Dach1 activated this reporter even in the presence of Dach1. (G) By contrast, co-expression of DD2 did not activate this promoter, but instead cancelled the Dach1-mediated repression.