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Fig. 4. Smads bind to multiple sites in the C15 enhancer.
(A) Schematic representation of the DNA fragments comprising the 650 bp
enhancer that were labeled for mobility shift assays with GST-Smad proteins.
Fragments that formed complexes are highlighted in blue. (B) DNA
sequence of wild-type and mutated oligonucleotides containing the putative
Smad sites from the C15 enhancer and one from the Race
enhancer (R42) that were used for mobility shift assays and in vitro
mutagenesis. Putative Smad sites are highlighted in blue, mutated sequences in
green. The different types of Smad sites are denoted above the sequences: GC,
GC-rich; SBE, Smad-binding element; DSC, Drosophila Smad consensus.
(C) Gel of GST-Mad incubated with the labeled wild-type
oligonucleotides. The first lane in each section contains free probe. The
second and third lanes contain increasing amounts of GST-Mad (10 and 30 ng;
indicated by blue steps). (D) Gel of GST-Mad incubated with wild-type
(bold) or mutated oligonucleotides. Lanes 1-6, M2 and mutations in one of the
four GC-rich sites (denoted 1-4), or all four sites (m). Lanes 7-12, M5 and
mutations in the GC (gc), the SBE (sb) or either half of the DSC (ds1, 2), or
all of the sites (m). Lanes 13-16, M6, M7 and mutations in all putative sites
(m). (E) Summary of Smad-(blue ovals) and Zen-(red ovals) binding sites
in the 650 bp enhancer (thick black line). Relative locations of the
oligonucleotides and the 350 bp enhancers are delineated by thin horizontal
lines.
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