Figure 2.

Comparison of selectivity profile for PK105b and Cy5-V-DPP. (A) Recombinant serine proteases (neutrophil elastase, proteinase 3, or trypsin) were incubated with increasing concentrations of Cy5-V-DPP or PK105b and binding was assessed by in-gel fluorescence. (B) Kinetic measurements of recombinant neutrophil elastase activity using the fluorogenic substrate probe, AAPV-AMC in the presence of DMSO (vehicle control) or the indicated concentration of PK105b, Cy5-V-DPP or Sivelestat. Error bars are shown as mean ± SEM. (C,E) Lysates from mouse bone marrow or pancreas were incubated with increasing concentrations of Cy5-V-DPP or PK105b and binding was assessed by in-gel fluorescence. (D,F) PK105b-labeled lysates were immunoprecipitated with antibodies for neutrophil elastase, pancreatic elastase (PE) or trypsin 3 (Try3), as indicated. In (F), gain settings for pulldowns were enhanced in order to observe faint bands (right panel). Note: Cy5-V-DPP and PK105b labeling are depicted at equal gain settings, set independently for each sample type.