Western blot analysis of PI3K/AKT and RAS/ERK pathways

One million HeLa cells were seeded in 60-mm petri dish format for 24 hours prior to the transfection. Cells were transfected with a total of 0.9 μg/ml of four different mixtures of the empty vector and MYO1C-construct, as described earlier, to produce final concentrations of MYO1C-construct (0.0, 0.3, 0.6 or 0.9 μg/ml) to investigate potential MYO1C dose-dependent effects in the present experimental setup. Cell lysates were prepared at 24 hours post transfection and subjected to Western blot analysis for MYO1C protein as well as 10 key components of the PI3K/AKT pathway (AKT, pAKT_S473, pAKT_T308, IRS-1, PTEN, 14-3-3β, p85, p110α, p110β, p110δ, Table 1) and three downstream proteins of the RAS/ERK signaling pathway (RAS, ERK and pERK). Information on the manufacturers from which the antibody orders were made as well as antibody dilutions used in the experiments are presented in Table 1.