Transfection of Cells with Akt Kinase

1.5×10⁶ cells prepared as described above were re-suspended in 100 μl of nucleofector solution (Amaxa Mouse Neuron Nucleofector kit; LONZA). Cells were co-transfected with 0.75 μg of an expression vector encoding GFP (pmax-GFP; LONZA) to mark transfected cells and 2.25 μg of either pcDNA3 (empty vector control) or a construct encoding constitutively active Akt kinase (Akt-CA, full length mouse Akt1 with the 14-amino acid src myristoylation signal at its NH₂ terminus [24 (link)]) or a construct encoding a dominant negative Akt mutant lacking kinase activity (Akt-DN, human Akt1 inactivated by a Lys179 to Ala mutation [25 (link)]). The Akt constructs were kindly provided by S. Pons (Biomedicine Institute, CSIC, Barcelona, Spain). Transfections were performed in an Amaxa transfection device (Amaxa Biosystems), using program G-013 according to the manufacturers specifications. Cells were plated and grown on poly-L-lysine/laminin coated coverslips as described above.