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. 2017 Feb 8;7:41871. doi: 10.1038/srep41871

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Copyright © 2017, The Author(s)

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(a) Western blot analysis of conditioned medium (Med) and cell lysate (Lys) of HEK293F cells co-transfected with ADAMTS17^EA (17-EA) and ADAMTS17 (17), ADAMTS17^EA (17-EA), ADAMTS17-PCD (PCD), or empty vector (v) indicate trans-cleavage of ADAMTS17^EA by ADAMTS17 and ADAMTS17-PCD. White arrows indicate lanes with cleavage products, detected with the anti-propeptide antibody (anti-PP, green). Anti-myc (red) detects zymogen and mature forms of ADAMTS17 in the cell lysates and zymogen and mature form of ADAMTS17-PCD in the cell lysate and conditioned medium. (b) Western blot analysis of conditioned medium (Med) from HEK293F cells, stably expressing ADAMTS17-AD (AD) and transiently expressing ADAMTS17 (17), ADAMTS17^EA (17-EA), ADAMTS17^RA (17-RA) or empty vector (v). Note reduced levels of ADAMTS17-AD in the presence of ADAMTS17 or ADAMTS17^RA (left-hand panel, red) and mass shift of furin-resistant species compared to wild type (brackets indicated by Δ1 and Δ2) (middle panel, green). The proteolytic product detected with anti-AD is indicated with an arrow (right-hand panel, red) and only appears upon transfection of ADAMTS17-AD expressing cells with ADAMTS17 or ADAMTS17^RA. (c) Western blot analysis of conditioned medium (Med) from HEK293F cells, stably expressing ADAMTS17-AD (AD) and transiently expressing ADAMTS17-PCD (PCD), ADAMTS17-PCD^EA (PCD-EA), ADAMTS17-PCD^RA (PCD-RA) or empty vector (v). The mature form of ADAMTS17-PCD (PCD-M) was not generated in ADAMTS17-PCD^RA, indicating lack of furin processing (left-hand panel, red and middle panel, green). Furin processing did not abolish proteolytic activity as observed by reduction in the intensity of anti-myc reactive ADAMTS17-AD (left-hand panel, top band) or the appearance of an additional band reactive with the anti-AD antibody (arrow, right panel, red). Note that the α-Myc antibody (red) detects both ADAMTS17-PCD and ADAMTS17-AD. M, mature enzyme; Z, zymogen.