Fig. 7. N-terminal coiled-coil domain of mVps2 is required for the formation of E235Q compartments but not for its binding to SKD1. (A) Single and co-transfection of full-length mVps2-myc (a-c), N-mVps2-myc (d-f) and C-mVps2-myc (g-i) with wild type (b, e and h) or SKD1(E235Q) (c, f and i) in HeLa cells. The transfected cells were subjected to immunofluorescence microscopy with anti-myc and anti-SKD1 antibodies. The distribution of single transfected myc-tagged mVps2 variants is indicated in black and white images (a, d and g), while the merged images from the double transfected cells show the localization of myc-tagged mVps2 variants (red) and SKD1 (green). Scale bar: 20 µm. (B) Proportion of cells with E235Q compartments among those double transfected with SKD1(E235Q) and one of the mVps2 variants, tagged with either GFP (green bar) or myc (blue bar). Mean±s.d. values of 3-5 independent experiments are shown. (C) GST pull-down analyses of mVps2-GFP chimeras. Top panel shows immunoblots of cell lysates prepared from HeLa cells transiently expressed full-length mVps2-GFP (lane 1), C-mVps2-GFP (lane 2) and N-mVps2-GFP (lane 3). The cell lysates were incubated with GST-SKD1 (middle panel) or GST alone (bottom panel) adsorbed to glutathione-Sepharose beads. The bound proteins were eluted and immunoblotted with anti-GFP antibody. Asterisks indicated the GFP fusion protein bands of the predicted sizes, 61, 57 and 55 kDa, respectively.