DrrA-Mediated Tethering Assay

HEK293-FcγRII cells grown on poly-l-lysine–coated 24-well plates (10⁵ cells/well) were permeabilized as described previously (Arasaki et al., 2012 (link)). Permeabilized cells were incubated with or without 3 µg purified His-DrrA for 1 h at room temperature. Cells were washed, and 100 µl of a PNS fraction from HEK293-FcγRII cells was added with GTP at 0.5 mM final concentration. Cells were incubated for 1 h at room temperature and then washed extensively. To assay tethering, 100 µl lysis buffer (100 mM NaCl, 1 mM MgCl₂, 20 mM Hepes-KOH, pH 7.2, 1% Triton X-100, and protease inhibitors) was added to the cells to liberate Luciferase-KDEL from vesicles, and activity was measured using a Luciferase assay kit (New England Biolabs).