Functional assay to determine physiological relevance of LF-mediated enhancement of CatG activity.

<p>A) Granulocytes were treated with PMA, Zymosan, or kept in medium (control). The granulocyte-derived supernatant was incubated with MARS116 and CatG activity was detected after SDS-PAGE and streptavidin-HRP blot. LF was visualized by LF-specific immunoblot (left panel). The CatG band intensity was quantified by ImageJ and summarized (right panel). One representative out of two independent experiments is shown. B) Human blood from buffy coats were incubated with CatG (8, 4, 2, or 1 μg/ml), with or without LF (250 μg/ml, 3.27 μM), CatG with CatG inhibitor (10 μM) and LF, or CatG and LF with DMSO for 30 min at RT. Platelet activation was determined by the increase of cell surface levels of CD62P analyzed by flow cytometry (n = 5 different donors; for CatG+LF+CatGinh. and CatG+LF+DMSO, n = 3).</p

Detection of CatG activity under the control of LF or heparin.

<p>800 ng/ml CatG was incubated with or without LF or heparin (250 μg/ml) and MARS116 for 0 h or 2 h. CatG activity was detected via SDS-PAGE and streptavidin-HRP blot (upper panel). One representative out of two independent experiments is shown. The intensity of the respective bands were determined by ImageJ (lower panel).</p

Analysis of pH dependent enhancement of CatG activity.

<p>CatG (800 ng/ml) or both LF (250 μg/ml, 3.27 μM) and CatG were incubated with MARS116 for the indicated time points under A) pH 5 or B) pH 7.4 conditions at 37°C. Samples were resolved by SDS-PAGE, transferred to PVDF membrane, and visualized by streptavidin-HRP. C) CatG and MARS116 were independently preincubated for 0, 30, or 60 min. After 60 min, in one set of samples LF was added and further incubated. The band intensity was analyzed by ImageJ and summarized (right panel). Three independent experiments were performed.</p

Use of different activity-based probes to visualize CatG activity.

<p>A) The structure of MARS116, MARS123, and MARS125. B) 800 ng/ml of CatG, NE, or PR3 with or without 250 μg/ml (3.27 μM) LF were quantified by using MARS116, MARS123, or MARS125. One representative out of three independent experiments is shown (left panel). The CatG band intensity was quantified and summarized in the bar diagram (right panel).</p

Degradation of LF.

<p>Recombinant human LF (50 μg/ml) was incubated with CatG (50 μg/ml) or CatS (5 μg/ml) at pH 7.4 and CatL (5 μg/ml) at pH 5.0 for 6 h at 37°C. The degradation products were visualized by SDS-PAGE and Coomassie staining. A representative result of three independent experiments is shown.</p

Determine of cathepsin G (CatG) activity in the presence of lactoferrin (LF).

<p>A) 800 ng/ml of purified human CatG were incubated with indicated amounts of LF together with the activity-based probe MARS116 for 1 h at RT. MARS116 binds covalently to the active site of CatG. The CatG-MARS116 complex was resolved by SDS-PAGE, transferred to PVDF membrane, and visualized by streptavidin-HRP (left panel). The density of the respective bands were quantified by ImageJ and summarized in the bar diagram (right panel), n = 5. B) 800 ng/ml (28.07 nM) of CatG, CatG with 250 μg/ml (3.28 μM) LF, CatG preincubated with CatG inhibitor (10μM), CatG preincubated with CatG inhibitor and then added LF, or LF were incubated with MARS116 for 1 h at RT. LF 3.28 μM:CatG 28.07 nM ratio = 116.85. Two independent experiments. C) CatG activity was analyzed by using the colorimetric substrate Suc-VPF-pNA. 800 ng/ml CatG, CatG with CatG inhibitor (10 μM), LF (250 μg/ml, 3.27 μM), CatG with CatG inhibitor and LF, or LF were preincubated for 15 min at RT. Afterwards, the Suc-VPF-pNA was added and the measurements were performed in duplicates. One representative diagram is shown (two independent experiments, n = 2).</p