Large-scale screening assay to measure epidermal growth factor internalization.
Recently, we showed that the internalization of the epidermal growth factor (EGF) receptor is inhibited by hydrogen peroxide (H(2)O(2)) in human fibroblasts. In order to test the effect of various stress conditions on receptor internalization and to test a variety of antioxidants in their capacity to prevent or reduce the H(2)O(2)-induced inhibition of internalization, a screening assay was developed to measure the internalization in 96-well plates. In this assay, cells are exposed to biotin-conjugated EGF and the amount of internalized EGF is detected with horseradish peroxidase-conjugated streptavidin. We show that the results obtained by this new assay are comparable with those from internalization studies performed with radioactive labeled EGF. Therefore, the cellular internalization assay as presented here is a reliable method to measure EGF receptor internalization. Moreover, because elaborate processing of the cells is not required, the assay is a relatively fast and inexpensive method to study ligand-induced internalization in 96-well plates and thereby is suitable for large-scale screening of compounds or conditions interfering with this internalization.
de Wit
R
R
Department of Molecular Cell Biology, Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands. R.deWit@bio.uu.nl
Hendrix
C M
CM
Boonstra
J
J
Verkleu
A J
AJ
Post
J A
JA
D016475:3T3 Cells; D000818:Animals; D004705:Endocytosis; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D051379:Mice
D004815:Epidermal Growth Factor; D006861:Hydrogen Peroxide; D066246:ErbB Receptors