Figure 3

Functional assays. (A) Partially purified RECK∆C incubated for up to 8 days at 37 °C with or without o-phenanthroline (o-Phe.) or an inhibitor cocktail (cOmplete EDTA-free). (B) Degradation of fibrinogen. Lane 1, intact fibrinogen; lane 2, control, fibrinogen incubated with partially purified RECKΔC for two days shows degradation; lanes 3–9, same except for a previous one hour-incubation of RECKΔC with AEBSF, inhibitor cocktail, marimastat, batimastat, EDTA, PMSF or o-phenanthroline. Fibrinogen cleavage does not occur with similarly purified RECKΔC from (C) S2 cells or (D) Expi-CHO cells (both, left lane, fibrinogen alone; right lane, fibrinogen plus RECKΔC [black arrow]). (E) Incubation of fibrinogen (lane 1, control) with SEC fractions containing only FRAG-1 (22–25) show no degradation. However, the substrate is cleaved by a coeluting MMP-14 CD contamination (fraction 26). (F) Incubation of fibrinogen (lane 1) with partially purified N-TES without (lane 2, control) or with (lanes 3–7) inhibitors. (G) An N-TES preparation purified by SEC cleaved fibrinogen (control). This cleavage was abolished with AEBSF. Figure panels with lanes/parts from different gels/blots show white separation lines. All original gels can be found in the supplementary materials.