FIG. 4.

Preferential generation of PI-3,4,5-P₃ in association with actin structures detected by low-level expression of GFP-PH-GRP. L6 myoblasts were transiently transfected with 0.5 μg of GFP-PH-GRP1 cDNA for 16 h. Cells were serum deprived (4 h) and then stimulated with 100 nM insulin for 10 min at 37°C. After this period, the cells were fixed, permeabilized, and stained for F-actin (rhodamine-phalloidin). For the basal condition, images from both the ventral focal plane (near the bottom of the cell) (a and b) and the dorsal plane (near the top of the cell) (c and d) were obtained using a confocal microscope. For insulin-stimulated cells, images from the dorsal focal planes were obtained (e and f). Closed arrowheads indicate regions of remodeled actin filaments and localization of GFP-PH-GRP1 protein in insulin-stimulated cells. The images are representative of at least four experiments. Bar, 10 μm.