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. 2012 Oct 17;7(10):e47793. doi: 10.1371/journal.pone.0047793

Figure 2. ADAMTS-4 cleaves Reelin at both the N- and C-terminus.

Figure 2

(A–D, F) Anti-Reelin (G10, N-terminal antibody) immunoblots (IB). (A,B) Samples from Reelin-expressing HEK293 cells were collected after 12 hours incubation. (A) Metal chelator (PO, 50 µM), catechins (EGCG, 150 µM; ECG, 150 µM), and picetanol (PIC, 60 µM) inhibit Reelin cleavage. (B) Expression of TIMP-3, -1, and α2M, but not TIMP-4, inhibit Reelin cleavage. (C) Recombinant ADAMTS-4 (10 ng/µl) processes Reelin at both cleavage sites. Short vertical line at the bottom of the blot denotes that the last lanes, from the same blot, were joined for visual presentation. (D) Recombinant TIMP-3 (1.5, 3, 6, 12 ng/µl) and α2M (44, 72, 88,100 ng/µl) inhibit the proteolytic action of ADAMTS-4 on full-length Reelin. (E) Anti-ADAMTS-4 immunoblots (IB) showing reduction of ADAMTS-4 protein levels in HeLa cells transfected with two different ADAMTS-4 shRNA constructs, but not with scrambled shRNA nor with empty vector. (F) ADAMTS-4 shRNA transiently inhibited Reelin cleavage in Reelin-expressing HeLa cells. The frame emcompasses overexposed lanes from the blot on the right (asterisks) for better visualization of the Reelin fragments.