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Fig. 6. Smad and homeodomain consensus sites are necessary for BMP responsiveness
of Msx2 transgenes. (A) Smad or homeodomain consensus sites (bold)
were mutated in the 52 bp element as shown above and below the sequence (red).
Potential Brinker (blue) and Smad4 (green) sites are identified. (B-D) EMSA
was used to assess the effects of the mutations on binding of bacterially
expressed Smad4 MH1 domain (B), and in vitro transcribed/translated Prx1b, a
paired-class homeoprotein (D). Amounts of GST-Smad4 protein used were 50, 250
and 500ng (wild-type control; 500 ng GST). The relative amounts of Prx1b
lysate used were 1, 2, 4 and 10 µl (controls; 20 µl). Band intensities
of the faster-migrating, primary Smad4-DNA complexes are plotted in C. (E-J)
Effect of Smad site and homeodomain site mutations on
52bpMsx2-hsplacZ transgene expression in transgenic embryos at E11.5
(E-G), and BMP responsiveness in limbs (H-J). (K-O) We introduced the
homeodomain site mutation into the 560 bp fragment
(Fig. 1A,
Fig. 3A) and examined the
expression and BMP responsiveness of this construct in 10T1/2 cells (K) and in
transgenic embryos (L-O) at E11.5. Embryos in E-G,M were stained for
lacZ activity overnight, while that in L was stained for 1 hour.
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