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jws-dvdy.21035.tif8255K Supplementary Figure 1 Duf, Rols, Sns, and Blow localize in Hek293 cells in the same way as they do in C2C12 cells. The actin cytoskeleton is labeled with Phalloidin-TRITC (red in B, C, E, F, Q, and R), the expressed proteins detected using antibodies as indicated. The nuclei of the cells are stained with H?chst (Blue in C, F, I, L, O and U). (A-C) Hek293 cells that are transfected with Duf change morphology and form long, thin filopodia that seem to release small Duf-positive vesicles (arrows in A and A?). (D-F) When Hek293 cells are transfected with a HA-tagged Rols construct, the protein can be detected in a dot-like pattern throughout the cytoplasm (arrow in F?). (G-I) In contrast, cotransfection with Rols and Duf leads to localization of Rols at the cell membrane (arrows in G and I?). (J-L) Sns does not localize at the membrane of Hek293 cells either when transfected alone or together with Blow. Nevertheless, Sns can be detected in the filopodia of the cells (arrow in K and K?). (M-O) When cells that are transfected with Sns and Blow are mixed with Duf- and Rols-expressing cells, Sns can be found at the membrane (arrow in M and M?). (P-R) In cells that are transfected with Blow, colocalization with the actin cytoskeleton can be observed as visualized by Phalloidin staining especially at the points where the cells form protrusions (arrow in P and R?). (S-U) When a Duf/Rols-expressing cell (marked with an asterix in T and U?) is in contact with a Sns/Blow-expressing cell (marked with an arrowhead in T and U?), the proteins do not concentrate at the points of cell-cell contact (arrow in S and U?).

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