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Figure 3 Plasma SCUBE1 as Proteolytic Fragments
(A) Two-dimensional electrophoresis and Western blot of a representative plasma sample of acute coronary syndromes patients probed with antisignal peptide–complement proteins C1r/C1s, Uegf, and Bmp1–epidermal growth factor-like domain containing protein 1 (SCUBE1) antibody. Two spots (arrowheads) represent the proteolytic fragments (MW = 95 kDa, pI = 5 and MW = 80 kDa, pI = 6.5, respectively), different from the full-length SCUBE1 protein (MW = 135 kDa, pI = 6.7, arrow). These 2 fragments have been seen in multiple acute coronary syndromes plasma samples. (B) The secreted SCUBE1 protein is cleaved in the presence of fetal bovine serum (FBS). The recombinant Flag.SCUBE1 protein was produced in HEK-293T cells in the presence or absence of FBS. Immunoprecipitation of Flag.SCUBE1 was followed by Western blotting using anti-FLAG antibody. (C) Proteolytic cleavage within the spacer region of the secreted SCUBE1. Recombinant Flag.SCUBE1 full-length (FL), deletion mutants (D1 and D2) protein was compared with the cleaved SCUBE1 fragment. The positions of FL protein (arrow) or its cleaved fragments (arrowhead) are indicated. (D) Domains of the SCUBE1 full-length and deletion mutants. Arrow shows a putative protease cleavage site (RXXR) within the spacer region. "Y" indicates potential N-linked glycosylation site. CUB = complement proteins C1r/C1s, Uegf, and Bmp1 domain; E = EGF-like repeats; SP = signal peptide.
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