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. 2019 Jan 31;18(5):818–836. doi: 10.1074/mcp.RA118.001304

Fig. 2.

Fig. 2.

KLK7-mediated cleavage of thrombospondin 1. A, Peptograph (replicate 1) representing KLK7-mediated hydrolysis of THBS1 in SKOV-3 cell CM See supplemental Fig. S2 for peptograph description. Arrowheads to the right indicate the migration of THBS1 retrieved from the control (open) and KLK7-generated fragments of THBS1 (filled). The higher molecular weight (MW) peptides (blue and gray) are abundant in gel slices 1–4 (Log2 KLK7/control = 0 to −5), representing the fragments derived from the full-length protein identified in both KLK7- and buffer-treated samples. The lower MW fragments are abundant in gel slices 5–15 (Log2 KLK7/control = 0–5), representing the KLK7 cleavage fragments (red) found in the KLK7-treated sample. A schematic of selected protein domains, based on annotation in the UniProtKB, is shown beneath the X-axis (purple boxes), aligned with the appropriate residues. H, heparin-binding; V, von Willebrand factor, type-C; THBS1/3, THBS type-1/3 repeat; E, epidermal growth factor-like; THBS C-, THBS C-terminal. The TAILS identified KLK7 cleavage sites C-terminal to Y258 and Y665 are shown by red dotted vertical lines and B, represents the respective spectrums. C, Peptides identified in the qPROTOMAP analysis (PEP, posterior error probability; Score, The sum of the ion scores of all peptides identified; PSMs, peptide spectrum matches). D, Silver-stained 12% SDS-PAGE showing hydrolysis of recombinant (r) THBS1 (500 ng) by recombinant active KLK7 (1/10 −1/1000 molar ratio to rTHBS1); buffer and dmKLK7 treatments were used as controls. Arrow heads in colors depict, yellow: THBS1 full length protein; white: cleavage fragment ∼130 kDa; red: 28 kDa cleavage fragment; black: KLK7 or dmKLK7. Western blot analysis of KLK7-treated rTHBS1 using antibodies targeting E, full length and F, N-terminal THBS1 confirmed the KLK7-mediated generation of N-terminal heparin binding domain containing fragment. Arrowheads indicate different protein products: yellow: full length THBS1, 150 kDa; white: 130 kDa; green: 100 kDa; gray: 90 kDa; red: 28 and 25 kDa fragments. The MW of the protein standard (kDa) is indicated to the left.