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Fig. 3. E-like overexpression leads to microtubule depolymerization and Golgi membrane fragmentation. (A) Double-label immunofluorescence of HeLa cells transfected with EGFP alone (a,b), E-like-EGFP (c,d) or E-like-EGFP followed by a brief incubation with nocodazole (e,f). An anti--tubulin antibody recognized by a Texas Red-conjugated secondary antibody was used for the detection of microtubules and tubulin. Arrows in panels c and e highlight MT destruction in cells overexpressing E-like. (B) HeLa cells transfected with E-like-EGFP (El-EGFP) and visualized with a Texas Red-conjugated antibody recognizing an anti-ß-cop antibody to detect Golgi stacks. Golgi membranes in transfected cells are arrowed. (C) E-like is not a microtubule binding protein. ³⁵S-labeled in vitro translated hGEF-H1 (GEF) and E-like (El) were incubated in the presence of taxol-stabilized microtubules and sedimented through a sucrose cushion. Pellet (P) and supernatant (S) fractions containing equal amounts of protein were resolved by SDS-PAGE. Equivalent aliquots of the starting material are shown in the first two lanes. Molecular mass standards are shown on the left. Bar, 10 µm.

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