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Fig. 8. N::EYFP inhibited Fmi-dependent cell aggregation and might have interacted with Fmi in cultured cells. (A-F) S2 cells were transfected with a plasmid without insert (A) or with one of plasmids encoding various Fmi forms (B-F), together with an EGFP plasmid; and they were examined to see whether they formed aggregates or not. Cells that expressed Fmi (B), Fmi::EYFP (C), or Ctail (D) assembled, but those that expressed CR::EYFP (E) or N::EYFP (F) did not. (G,H) Coexpression of N::EYFP with Fmi inhibited Fmi-dependent cell aggregate formation. S2 cells were co-transfected with the EGFP plasmid and the Fmi expression plasmid, together with one without insert (G) or with the N::EYFP plasmid (H). (I) Fmi and HA-N::EYFP were expressed in HEK293T cells. The cell lysate (lane 1) and immunoprecipitates (IP) obtained with either anti-myc (negative control, lane 2), anti-HA (lane 3), or anti-Fmi (lane 4) were blotted with anti-Fmi antibodies. The arrowhead points to Fmi molecules. Fmi was coimmunoprecipitated with HA-N::EYFP (lane 3). (J,K) S2 cells were transfected with a plasmid expressing (J) membrane-bound Venus (Venus-pm) or with (K) N::EYFP plasmid, together with the Fmi expression plasmid. Cells were spread on ConA-coated dishes and stained for GFP (left panels; green in merged right panels) and Fmi (middle panels; magenta in merged right panels). We used 0.05% saponin to permeabilize plasma membranes in order to preserve intracellular vesicular structures. Over 20 cells were observed for each transfection experiment and all cells showed similar protein distributions to cells shown in J and K.

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