A431 cells express approximately 10e6 epidermal growth factor (EGF) receptors at the cell surface. Upon stimulation with EGF, A431 cells exhibit a dramatic increase in phosphorylation of EGF receptors followed by activation of major cell signaling pathways, such as PKB/Akt and MAP kinase pathways. Downstream of these cell signaling pathways, a variety of cytoskeletal, cytoplasmic, and nuclear proteins become phosphorylated at different time points after EGF stimulation. Confluent cultures of A431 cells were serum starved overnight. Cells were then either left untreated or treated with human EGF (100 ng/ml) for 30 minutes at 37°C. Cells were lysed in 1% SDS, 1.0mM sodium ortho-vanadate, 10mM Tris (pH 7.4) buffer. Protein concentration was determined using the BCA method (Pierce) before diluting to final concentration and buffer.