FIG. 7.

High-level expression of GFP-PH-GRP1 or wortmannin pretreatment inhibits insulin-stimulated actin remodeling. L6 myoblasts transiently transfected with 0.5 μg of GFP-PH-GRP1 (A) or GFP-PH(K273A)-GRP1 (B) or GFP-PH-Akt (C) cDNA were stimulated with (Ac to h; Ba and b; and Ca to d) or without (Aa and b) 100 nM insulin for 10 min at 37°C. L6 myoblasts transfected with GFP-PH-GRP1 were also exposed to 100 nM wortmannin for 20 min prior to and during insulin stimulation (A, g and h). After this period, the cells were fixed, permeabilized, and stained for F-actin (rhodamine-phalloidin). Arrowheads indicate regions of remodeled actin filaments and localization of GFP-tagged protein in insulin-stimulated cells. Cells highly expressing the constructs are outlined. The images are representative of three experiments. Bar, 10 μm.