Figure 1

(A) Expression and activation of ectopically expressed farnesylated Akt1 in NCI H460 cells. Control transfected NCI H460 cells or cells expressing farnesylated Akt1 were serum starved overnight (-) and then stimulated for 15 min with medium containing 10% FCS (S) or 10% FCS plus a cocktail of 10 ng ml⁻¹ each EGF, IGF-1, and PDGF (GF). Cell lysates were analysed by immunoblotting with antibodies specific for Akt1, FLAG-epitope, phospho-Akt (Thr 308) or phospho-Akt (Ser 473) as indicated. The ectopically expressed farnesylated Akt1 devoid of the PH domain migrates at an MW of approximately 50 kDa. (B) Akt kinase activity in NCI H460-Akt1 cells and in control cells. Cells were serum starved for 16 h and then stimulated as described above. Endogenous Akt1 from control transfected cells and endogenous Akt1 plus farnesylated Akt1 from NCI H460-Akt1 cells were immunoprecipitated with an Akt1 specific antibody. Immunoprecipitates were used in kinase assay reactions with GSK-3-fusion protein as substrate. Immunoblots of the reactions were assayed with antibodies specific for phospho-GSK (Ser 21/9) (lower panel) and Akt1 (upper panel). (C) Proliferation rates of NCI H460 control transfectants and NCI H460-Akt1 cells. 3 × 10⁴ cells (as indicated) per well were seeded into six wells into a medium containing 0.5% FCS. At the indicated time points, the cells were harvested by trypsinisation and cell numbers were determined using a Coulter counter. Each sample was determined in triplicates. Two independent NCI H460-Akt1 cell clones were analysed. (D) Expression analysis of p21^Waf1 and p27^Kip1 in NCI H460-Akt1 cells vs control cells. Cell lysates were analysed by immunoblotting with antibodies specific for p21^Waf1, p27^Kip1, and β-actin as a loading control.