Detection of EGF-induced EGF receptor degradation and tyrosine phosphorylation in intact cells.

In this work, a simple, sensitive, and non-isotopic assay system for the detection of EGF-induced EGF receptor degradation and tyrosine phosphorylation in intact cells is described. In this system, boiling Laemmli sample buffer was directly added to cultured Chang liver cells to stop the reactions in the cells stimulated by EGF and to make whole-cell extracts. The effects of EGF concentration and incubation time on the EGF-induced degradation and tyrosine phosphorylation of EGF receptor were successfully determined using monoclonal anti-EGF receptor, recombinant anti-phosphotyrosine peroxidase conjugate, and enhanced chemiluminescence (ECL) Western blotting assay system. Unlike other assay systems, the use of radioisotopes was avoided in this determination. The assay system is linear up to 100 micrograms sample protein from whole-cell extracts for the detection of EGF receptor and EGF-induced autophosphorylation. This assay may be easily adopted for identification of other growth factor receptors and phosphotyrosine-containing proteins in intact cells, using appropriate anti-growth factor receptor antibodies.