The t-SNAREs syntaxin4 and SNAP-23 become concentrated in the insulin-induced membrane ruffles. Serum-deprived L6 GLUT4myc myotubes were stimulated with (d–f and j–l) or without (a–c and g–i) 100 nM insulin for 10 minutes at 37°C, followed by fixation and permeabilization. Actin was labeled with Oregon Green–conjugated phalloidin. Syntaxin4 (a–f) and SNAP-23 (g–l) were stained with specific polyclonal antibodies, followed by Cy3-conjugated secondary antibody as described in Methods. Composite images of F-actin and t-SNARE protein staining are presented in panels c, f, i, and l. Arrows indicate regions of remodeled actin filaments. The figure illustrates the diverse appearance of the dorsal actin structures depending on their three-dimensional nature and the direction of the optical section. The images are representative of three experiments. Scale bar = 10 μm.