Identification of the cleavage sites in sVEGFR-1 by MMP-7. (A) Human recombinant sVEGFR-1 was incubated with MMP-7 at different times in the original buffer to which 0.05% Brij-35 had been added, and analyzed by 7.5% SDS-PAGE and Western blotting. ▶ indicates the full-length recombinant sVEGFR-1 (96 kDa). → indicates the proteolytic fragments of recombinant sVEGFR1. (B) Human recombinant sVEGFR-1 (1.6 μg) was incubated with MMP-7 at a substrate/enzyme molar ratio 2:1 for 24 hours. The digestion products were analyzed by 12% SDS-PAGE and SYPRO Ruby staining. The arrowhead indicates the full-length recombinant sVEGFR-1 (96 kDa). → indicates the degradation products of sVEGFR-1. → represents MMP-7, whose mobility in SDS-PAGE in our condition was identical to the sample of full-length MMP-7 alone and whose sequence started from Tyr¹  of the active form of MMP-7 according to the NH₂-terminal sequence analysis (data not shown). (C) The estimated molecular mass and NH₂-terminal sequence of the proteolytic products of sVEGFR-1 observed in this study. The degradation samples (total of 3.6 μg sVEGFR-1 and 375 ng MMP-7) were subjected to 12% SDS-PAGE followed by transferring to PVDF membrane for SYPRO Ruby staining. The N-terminal amino acid sequences of the visualized bands were analyzed.