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.