tPA cleaves Reelin at its C-terminal cleavage site.
(A) Schematic representation of the Reelin fragments detected with G10 anti-Reelin antibody (left) and immunoblot (IB) of hippocampus lysate from a 3 month-old wild-type mouse, showing full-length Reelin (F, 460 kDa), C-terminal cleaved (C, 380 kDa), and N-terminal cleaved (N, 160 kDa) Reelin fragments (right). (B-F) Anti-Reelin IB (G10, N-terminal antibody). (B) 100 mM Furin Inhibitor I (FI-I) inhibits Reelin cleavage in HeLa cells expressing Reelin. After 12 hours incubation of FI-I or DMSO alone, medium was collected (0 h) and further incubated at 37°C to check for potential Reelin degradation. (C) Recombinant tPA (25 ng/µl) cleaves Reelin at its C-terminal cleavage site. Recombinant serpin E1 (12.5, 37.5, 100 ng/µl) inhibits tPA action. (D) Neither the metalloproteinase inhibitors TIMP-3 (40 ng/µl) or α2M (40 ng/µl), nor the trypsin inhibitors SBTI/Aprotinin (1000 and 300 ng/µl) affect Reelin cleavage by tPA (25 ng/µl). (E) Co-expression of tPA and its inhibitors in Reelin expressing HEK293 cells. Sample (medium) for IB was collected after 12 h incubation. (F) Cross-linking Reelin with BS3 (30 minutes on ice) revealed that C-terminal cleavage does not affect Reelin dimerization (asterisk). The additional band observed with C-terminally cleaved Reelin (two asterisks) may represent higher-order oligomers. Note that the top of the gel is marked with the horizontal line. Indicated hours represent incubation times. All IB blots are representatives of three independent experiments.