Fig. 6

Characterization of the MACIT protein COL-99 of C. elegans. a Schematic structure of the COL-99 and COL-99::EGFP::FLAG polypeptides. The domain structure is presented as in Fig. 1 and cysteines (c) are also indicated. The regions used as antigens for the antibodies AB5625.11 and AB693 are marked. The C-terminal tag of COL-99::EGFP::FLAG is shown as a green circle. b Western blot analysis of subcellular localization of recombinant COL-99 in CHO cells. From the left to right: control lysate, total lysate of CHO cells expressing recombinant COL-99, concentrated medium sample, in the upper and lower panels detected with antibody AB5625.11 (upper) or AB693 (lower); and additional PBS-soluble proteins, and Triton X-100-extracted protein fraction from CHO cells expressing COL-99 in the lower panel only detected with AB693. c Immunofluorescent staining of CHO cells expressing COL-99. The signal was detected by antibody AB5625.11 and secondary anti-rabbit IgG conjugated with AlexaFluor 594. d Control staining with the secondary antibody only. Bars, 20 μm. e Inhibition of COL-99 ectodomain shedding by Furin Inhibitor I. Right lane, COL-99 expressing CHO cell cultures treated with 2 μM Furin Inhibitor I. Left lane, cells treated with an equivalent volume of methanol as solvent control. β-tubulin was used as a loading control in (b) and (e)