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Fig. 2. Response of stable ES reporter cell lines to BMP4. (A) Constructs used to generate transgenic ES cells. BRE-ß-galactosidase or BRE-luc reporter constructs and a PGK-hygromycin cassette were coelectroporated in wild-type ES cells. Single colonies were picked from selection medium (150 µg/ml hygromicin) and genotyped by PCR on genomic DNA. Transgenic ES cell lines were assayed for BMP-induced reporter expression. Basal reporter activity was observed in (B) unstimulated BRE-lac1 ES cells. (C) Reporter activity increased in response to 20 ng/ml BMP4. Bars, 50 µm (B,C). Activity of the reporter gene in ES lines BRE-lac1 (D), BRE-lac2 (E) and BRE-luc (F) increases with increasing concentrations of BMP4. Western blots for Id1 (anti-Id1 antibody) (G) and for activated Smad1/5/8 (PSmad1/5/8, PS1 antibody) (H) were performed on the same extracts as in D and E; expression profile of these proteins show good correlation with the reporter activity. Note that the middle band in the triplet corresponds to phosphorylated Smad1/5/8 (black arrowheads). 10 µg/ml of protein was loaded in each lane and confirmed by Ponceau S staining. Nonspecific bands are shown as loading control (l.c.). The results are represented as the mean of three independent experiments (mean±s.d.).
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