PI3K activity through the cell cycle. NIH3T3 cells were deprived of serum for approximately 30 hrs and injected with the PH-AKT-GFP plasmid. 15 hrs later cells were photographed under confocal microscopy either 20 min following serum stimulation (A), or without stimulation (B). Similarly, (C) cycling NIH3T3 cells were injected with the plasmid and photographed 15 hrs later. To confirm that the cycling cells retained the ability to stimulate PI3K activity, they were injected with the plasmid and deprived of serum for 0 hr (D), 5 hrs (E), 9 hrs (F) or 12 hrs (G) prior to addition of serum 20 min before confocal microscopy. Note the concentrations of fluorescence in cytoplasmic projections (E, F) or uniformly over the plasma membrane (G) following serum stimulation. The appearance of stimulated cells is clearly illustrated in a complete series of confocal sections at varying magnifications [presented in Additional files, movies 1, 2, 3].