Figure 3
ZnCl2 and CuCl2 inhibit HpHtrA activity in a concentration-dependent manner. (A) 5 µM FRET peptide were incubated with 250 nM HpHtrA and increasing concentrations of ZnCl2 (left panel) or CuCl2 (right panel) for 15 min (black bars) and 180 min (grey bars) at 37 °C in 50 mM HEPES buffer (pH 7.4). The data represent the relative fluorescence units (RFU) ± S.D. with fluorescent signals obtained from FRET peptide treated with HpHtrA wt set as 100%. Asterisks indicate statistically significant differences (****p < 0.0001; ns, non-significant). (B) 50 ng hCdh1 were incubated with 250 ng HpHtrA wt or inactive mutant (SA) and increasing concentrations of ZnCl2 (left panel) or CuCl2 (right panel) for 16 h at 37 °C in 50 mM HEPES buffer (pH 7.4). Full length hCdh1 (Cdh1FL) and cleavage fragments were detected by Western blot using an antibody recognizing the EC5 domain of hCdh1. HpHtrA and the auto-processed short HpHtrA (HpHtrAs) were detected using a polyclonal antibody. (C) 10 µg casein composed of αS1-, αS2- and β-casein were incubated with 250 ng HpHtrA wt or inactive mutant (SA) and with increasing concentrations of ZnCl2 (left panel) or CuCl2 (right panel). After incubation at 37 °C for 16 h proteins were separated by SDS-PAGE and visualized by staining with Coomassie Brilliant Blue G250.
