Figure 2

Dominant-negative SNARE domains impair lamellipodium extension. (A) CHO-K1 cells were transiently transfected with GFP-tagged constructs of the indicated SNAREs: GS15, VAMP4, VAMP3, syntaxin13 or SNAP23. Constructs encoded either the full length protein (FL) or the dominant-negative form (cyto; CΔ9 in the case of SNAP23). HeLa cells were transfected with a SNAP23 shRNA construct (SNAP23 shRNA). The cells were plated on FN-coated (20 μg/ml) coverslips and the extent of cell spreading was calculated as the increase in ventral surface area that occurred between 30 and 90 mins. The results are presented as percent of non-transfected control sample. More than 50 randomly selected transfected cells were analyzed per experiment. Means +/- SEM of at least three independent experiments are shown. (B) CHO cells were transfected as above, serum starved for 3 hrs and placed into a transwell chamber containing polycarbonate membranes coated on the underside with FN. Cells were allowed to migrate for 4.5 h, the membranes were fixed and the percentage of transfected cells migrated was assessed. Data is presented as the percentage of transfected cells migrated normalized to full-length controls and represent at least three independent experiments. (C) A sample of the same HeLa cells used in (A) were lysed and analyzed by Western blot for knockdown of SNAP23 expression. Actin served as a loading control. A set of representative blots are shown.