![Figure 1. CD63 and proNE form a complex. (A) A schematic diagram of the constructs used. The NE (preproNE) construct is shown with signal peptide, N-terminal dipropeptide (SE), mature enzyme sequence, and C-terminal propeptide. The full-length CD63 and the CD63 mutants; CD63ΔLEL, lacking the large extracellular loop (LEL), CD63Δ12 with deleted C-terminal lysosomal targeting motif, and CD63 dom neg (dominant-negative inhibition) with a single amino acid change in the C-terminal sorting motif are shown. N-terminal fusions of the CD63 variants with GFP were used for transfection of COS cells. The CD63 siRNA construct used is also shown with the complementary mRNA region forming a hairpin and marking the corresponding mRNA target region in the protein. (B) COS cells were transfected with plasmids carrying GFP-CD63 (CD63), NE, or GFP-CD63ΔLEL, as indicated. Cell lysates were examined by immunoblotting with anti-actin, anti-GFP (for detection of GFP-CD63 fusion proteins), and anti-cproNE, showing comparable expression of the plasmids. IP/Western was performed to examine coprecipitation between CD63 and proNE. Both GFP-CD63 and GFP-CD63ΔLEL coprecipitated proNE, as judged by IP with anti-GFP followed by Western blotting with anti-cproNE. In addition, proNE coprecipitated the GFP-CD63 and GFP-CD63ΔLEL, as judged by IP with anti-cproNE followed by immunoblotting with anti-GFP. (C) COS cells transfected as indicated were incubated for 24 hours with [35S]methionine/[35S]cysteine to achieve steady-state radiolabeling of proteins and then examined by IP and fluorography. A major radiolabeled 31-kDa proNE band was detected in cells expressing proNE or proNE plus GFP-CD63 (marked to the right with proNE). Upon coexpression, IP with anti-cproNE copurified proteins migrating as GFP-CD63 (●) or GFP-CD63ΔLEL (▲), and IP with anti-GFP copurified proteins migrating as proNE. In addition, GFP-CD63ΔLEL (▲) copurified proNE and vice versa. These data confirm complex formation between proNE and GFP-CD63 or GFP-CD63ΔLEL. The 85-kDa band of the supernatant corresponds to a covalent complex between proNE and α1-proteinase inhibitor.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/112/8/10.1182_blood-2007-10-116285/4/zh80200825580001.jpeg?Expires=1735837097&Signature=oQVUIbOLpl6Y-GnK1zP9Z~XGMUY2ei-NgCTR~aES3pVm-rAVWaXiQ62iPsjIv-xm9vcpb576TCd4jC~v2~DRiQ2OUF9kzMU80krNVf5HAJ-g9X078o~xYTQ~14srxf5bi8TNXsN69E7r13rcVqDRdDVlScQBQtx5eZ610TH2RyvqEvUF1gZo3NFcPBCPXoSOU3ncPWOyh89f4qAIcUOvEdPollFDXILe9tXEvPzv4ZC25payKkPdDPw4BBDkY0Y6hH6QgdpIBytS3YqTO6XcgeTbtYJqxMQ1dwxSpKu9sGMOcpQPiI-SzjwZURTMRg1tnGi7i7vlMvnH6bgBerR6WA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
CD63 and proNE form a complex. (A) A schematic diagram of the constructs used. The NE (preproNE) construct is shown with signal peptide, N-terminal dipropeptide (SE), mature enzyme sequence, and C-terminal propeptide. The full-length CD63 and the CD63 mutants; CD63ΔLEL, lacking the large extracellular loop (LEL), CD63Δ12 with deleted C-terminal lysosomal targeting motif, and CD63 dom neg (dominant-negative inhibition) with a single amino acid change in the C-terminal sorting motif are shown. N-terminal fusions of the CD63 variants with GFP were used for transfection of COS cells. The CD63 siRNA construct used is also shown with the complementary mRNA region forming a hairpin and marking the corresponding mRNA target region in the protein. (B) COS cells were transfected with plasmids carrying GFP-CD63 (CD63), NE, or GFP-CD63ΔLEL, as indicated. Cell lysates were examined by immunoblotting with anti-actin, anti-GFP (for detection of GFP-CD63 fusion proteins), and anti-cproNE, showing comparable expression of the plasmids. IP/Western was performed to examine coprecipitation between CD63 and proNE. Both GFP-CD63 and GFP-CD63ΔLEL coprecipitated proNE, as judged by IP with anti-GFP followed by Western blotting with anti-cproNE. In addition, proNE coprecipitated the GFP-CD63 and GFP-CD63ΔLEL, as judged by IP with anti-cproNE followed by immunoblotting with anti-GFP. (C) COS cells transfected as indicated were incubated for 24 hours with [35S]methionine/[35S]cysteine to achieve steady-state radiolabeling of proteins and then examined by IP and fluorography. A major radiolabeled 31-kDa proNE band was detected in cells expressing proNE or proNE plus GFP-CD63 (marked to the right with proNE). Upon coexpression, IP with anti-cproNE copurified proteins migrating as GFP-CD63 (●) or GFP-CD63ΔLEL (▲), and IP with anti-GFP copurified proteins migrating as proNE. In addition, GFP-CD63ΔLEL (▲) copurified proNE and vice versa. These data confirm complex formation between proNE and GFP-CD63 or GFP-CD63ΔLEL. The 85-kDa band of the supernatant corresponds to a covalent complex between proNE and α1-proteinase inhibitor.
