Fig. 5. Phospho-Akt is detected only in migrating cells. T98G cells were plated in the migration format onto laminin-coated slides (10 µg/ml) and allowed to migrate overnight. Cells were then incubated in serum-free media for 4 hours, treated with 100 ng/ml EGF or solvent control for 15 minutes then fixed. For treatment with the PI3-K inhibitor LY294002 (20 µM) was added 2 hours before time of EGF addition. Slides were then processed for total Akt (a and b) or phospho-Akt Ser 473 (c-h) immunocytochemistry as described in Materials and Methods. Arrows in d indicate more intense staining for phospho-Akt at the leading edge type of structures. Arrows in g indicate a different pattern of staining for phospho-Akt following EGF treatment. Bar, 5 µm.