Figure 7

Analysis of SNARE function in PMA-induced membrane ruffle formation. CHO-K1 cells were treated with 500 nM PMA in DMEM (PMA) or DMEM alone (control) for 10 mins, fixed, permeabilized and stained with anti-SNAP23 antibody, Alexa-488-conjugated secondary antibody (A, D) and rhodamine-phalloidin (B, E). A-C, control cell; D-F, PMA-treated cell. Some cross-reactive binding of the SNAP23 antibody in the nucleus is observed (A, D). C and F are overlays of panels A and B and D and E, respectively. Scale bar represents 10 μm. (G) CHO-K1 cells were transfected with GFP vector alone, GFP-tagged dominant-negative constructs of the indicated SNAREs, or GFP together with the light chain of tetanus toxin. HeLa cells were transfected for 72 h with GFP + PLOK-1 or GFP + vector containing SNAP23-targeting shRNA. Cells were treated with 500 nM PMA in DMEM (PMA) or DMEM alone (control) for 10 mins, fixed, permeabilized and stained with rhodamine-phalloidin. Samples were then imaged by confocal microscopy and actin-rich ruffles on the dorsal region of the cells were quantified as described previously. Ruffle indices (expressed as mean +/- SEM) are shown from three independent experiments (more than 25 randomly selected transfected cells were analyzed per condition in each experiment). (H and I) CHO-K1 cells were treated as above, lysed, and VAMP4 (H) or VAMP3 (I) were immunoprecipitated. Immunoprecipitates were washed and analyzed by SDS-PAGE-western blotting for SNAP23, upper blots. Then membranes were stripped and reprobed for the precipitated SNARE, lower blots.