A431 cells express approximately 106 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 (Cat.# AL9201) or treated with human EGF (100 ng/ml) for 5 minutes at 37°C (Cat.# AL9301). Cells were lysed in 1% SDS, 1.0 mM sodium ortho-vanadate, 10 mM Tris (pH 7.4) buffer. Protein concentration was determined using the BCA method (Pierce) before diluting to final concentration and buffer.
Western blot analysis of A431 cells (20 µg/lane) serum starved overnight and treated with EGF (100 ng/ml) for 5 min. The blot was probed with anti-paxillin (PM1071) or anti-paxilin (Ser-178).
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*All molecular weights (MW) are confirmed by comparison to Bio-Rad Rainbow Markers and to western blotmobilities of known proteins with similar MW.
This kit contains:
