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. 2021 Oct 21;297(5):101323. doi: 10.1016/j.jbc.2021.101323

Figure 1.

Figure 1

Expression of recombinant ADAMTS8.A, annotated ADAMTS8 domain structure. The zinc-binding sequence (residues 363–373) is shown below the cartoon. Histidine residues involved in zinc coordination are in bold, the active site glutamic acid residue is in red. B, expression of recombinant ADAMTS8 in HEK293T cells. Cells were transiently transfected using PEI. 4 h post-transfection, the cells were treated with either heparin (200 μg/ml)-containing medium or an equal volume of expression medium. Three days post-transfection, the conditioned medium was harvested and protein lysate extracted as reported in the Experimental procedures. Samples were analyzed by 4–12% reducing SDS-PAGE, followed by immunoblotting using an anti-FLAG antibody. C, immunoblot as in (B) but with approximately 4-fold higher protein content/lane to highlight additional bands. Red asterisks indicate major autolytic products. Z and M indicate the zymogen and mature metalloproteinase, respectively. Representative blots from n = 3 are presented. CR, cysteine-rich domain; Dis, disintegrin-like domain; Mp, metalloproteinase domain; Pro, prodomain; Sp, spacer domain; TSR, thrombospondin-like motif.