Human epidermoid carcinoma cell line, A431 cells that were 80-90% confluent were serum starved overnight, then exposed to 100 ng/ml EGF for 20 minutes at 37°C. Control cells were serum starved without the subsequent stimulation with EGF. Cell lysates were prepared according to the protocol. To use ab134005 Human EGFR Phosphorylation Antibody Array - Membrane, treated or untreated cell lysate was added into antibody array membrane. The antibody array membranes were washed and cocktail of biotinylated anti-EGFR was used to detect phosphorylated proteins on activated receptors. After incubation with HRP-Conjugated Streptavidin, the signals were visualized by chemiluminescence.