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Fig. 3. Binding of HtrA1 to various Tgfß family proteins. (A) Binding of HtrA1
and follistatin to Tgfß family proteins. GST pull-down assay was carried
out using GST-Tgfß fusion proteins and either HtrA1 (b) or myc-tagged
follistatin (a) in the presence 1.0 M NaCl. Bound HtrA1 or follistatin was
detected by western blotting. The righthand lane in each panel (asterisk) was
loaded with 3% input HtrA1 or follistatin. Lower panels show the recovered
GST-Tgfß fusion proteins stained with Coomassie blue. GST alone did not
bind to HtrA1 (left-hand lanes). (B) Binding of mutant HtrA1 proteins to Bmp4
or Gdf5. Results of a GST pull-down assay with 
FS (a), FS/PDZ
(b), linker/SP/PDZ (c) and S328A (d) are shown. One or two lanes on the
right were loaded with aliquots of input HtrA1 mutants; 10% (**) or
1% (*) of input in a and c, 20% (**) or 2%(*)
in b, and 15% (*) of input in d. Arrowheads indicate positions of
HtrA1 mutant proteins. (C) Solid phase binding assay of HtrA1. ELISA plate
wells coated with GST or GST-Bmp4 were incubated with 100 µl of a solution
containing myc-tagged HtrA1 protein (0.01-0.3 µg/ml). The binding of HtrA1
to GST (white triangle) or GST-Bmp4 (black square) was quantitated by
colorimetric assay using anti-myc antibody.
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