Figure 4. Recombinant HFt and LFt bind to HKa and and inhibit its anti-proliferative activity.

A. Binding of recombinant HFt and LFt to domain 5 of HKa. Purified and biotinylated recombinant HFt (20 µg) or LFt (20 µg) were incubated with 10 µg GST-D5 and the resulting complexes immunoprecipited with anti-GST antibody. Non-biotinylated HFt and LFt were used in the immunopreciptation shown in lane 4. The membranes were probed with streptavidin-HRP to detect biotinylated ferritin (b-ferritin) as well as with anti-GST antibody. B. Cells were treated with 50 nM HKa alone, or co-treated with 100 nM of HFt or LFt in the presence of 20 ng/ml bFGF and 10 µM ZnCl₂. Cell viability was assessed using an MTT assay 24 hours post-treatment. Shown are means and standard deviation of triplicate experiments with ** p<0.004. C. Cell lysates were collected 24 hours post-treatment and analyzed by western blotting. D. Phosphorylation of signaling molecules was quantified by densitometry. Shown are means and standard deviation of 3 independent experiments. *p<0.04; **p<0.002. b = biotinylated.