Fig. 1. Partitioning of MMP9 chimeras into distinct membrane domains. (A) Schematic representation of the MMP9-GPI and the MMP9-LDL chimeras. (B) Gelatinolytic activity in membrane (M) and cytosolic (C) fractions isolated from MMP9-GPI-, MMP9-LDL-, MMP9-wt and mock-transfected MCF-7 cells. ProMMP9 (solid arrowhead) and MMP9 (open arrowhead) activity in HT-1080 conditioned medium (H). (C) Gelatinolytic activity in conditioned medium from the same cells as in B after 24 hours of serum depletion. (D) MCF-7 cells expressing all MMP9 forms were fractionated in flotation gradients and analyzed by zymography. Fractions 1 and 6 represent the top and the bottom of the gradient, respectively. The same fractions were analyzed by western blot with anti-TfR and anti-VIP21 (CAV-1) as controls for non-raft- and raft-associated proteins, respectively. (E) Confocal microscopy of MMP9-expressing cells stained with cholera toxin ß-subunit (green) and anti-MMP9 antibody (red); yellow staining indicates colocalization of the molecules. The two-color overlays show representative cells for (a) MMP9-GPI, (b) MMP9-LDL and (c) MMP9-wt (n=50). The boxed regions in these images are enlarged in d, e and f, respectively. Scale bar, 10 µm. Single-color images are available: Fig. S1, http://jcs.biologists.org/supplemental/