Fig 2: Human WIF1 binds human Shh with high affinity. In pull-down assays, we have incubated rhWIF1 with rhShh, then the solution was mixed with anti-Shh (A) or anti-WIF1 antibodies (B). The mixtures were pipetted on Pierce Spin Columns filled with Pierce Protein A/G Plus Agarose. After washing, the bound proteins were eluted with Pierce Classic IP kit Elution Buffer (pH 2.8). Samples were analyzed by SDS/PAGE, and the proteins were visualized by Western blotting (WB) with specific antibodies against rhWIF1 and rhShh. In control experiments, we have incubated rhWIF1 with anti-Shh antibody and rhShh with anti-WIF1 antibody. After the affinity binding step samples were visualized by Western blotting with specific antibodies against rhWIF1 and rhShh. Lanes: input, sample applied to the column; unbound fraction; washing fractions, wash1, wash2; bound fraction. The numbers indicate the molecular mass values of proteins of the Page Ruler Plus Protein Ladder (MM).

Fig 3: The WIF domain of human WIF1 is a potent inhibitor of the signaling activity of human Shh. In the reporter assays monitoring the hedgehog antagonist activity of the WIF domain of WIF1 protein, Gli Reporter-NIH3T3 cells were grown to confluency, and after the removal of the culture medium, the cells were treated with 50 µL aliquots of 0.2 nM rhShh, preincubated for 5 min with 0–50 nM WIF-domain of WIF1 protein. The luminescence of cells treated with 0.2 nM Shh, 0 nM WIF was 5.88 ± 0.69 fold the luminescence of untreated cells. The figure shows the results of a representative experiment with four parallels. The data are expressed as percentage of the luminescence observed at 0 nM of WIF-domain.

Fig 4: Characterization of the interaction of human sonic hedgehog with human WIF1 by surface plasmon resonance spectroscopy. Various concentrations of rhShh (6.25 nM, 12.5 nM, 25 nM, 50 nM, 75 nM, and 100 nM) were injected over CM5 sensor chips containing immobilized rhWIF1 (at 0 s on the abscissa). For the sake of clarity, the concentrations of the Shh proteins injected over the sensorchips are not indicated; the SPR response increased parallel to the increase in Shh concentration.

Fig 5: Characterization of the interaction of the WIF domain of WIF1 with recombinant human sonic hedgehog by surface plasmon resonance assays. Various concentrations of rhShh (12.5 nM, 25 nM, 50 nM, 100 nM and 200 nM) were injected over CM5 sensor chips containing immobilized WIF domain (at 0 s on the abscissa). For the sake of clarity, the concentrations of the Shh proteins injected over the sensorchips are not indicated; the SPR response increased parallel to the increase in Shh concentration.