Measurement of protein tyrosine phosphorylation in cell adhesion.
This chapter describes biochemical, immunochemical, and microscopic approaches to measure protein tyrosine phosphorylation after cell adhesion. We have outlined detailed procedures to biochemically examine the phosphotyrosine content of cellular proteins by Western blotting, which in some cases can be performed using phospho-specific antibodies. Furthermore, we have described in detail the examination of subcellular localization of phosphotyrosine-containing proteins in focal adhesions using immunofluorescence. Finally, a quantitative fluorescence microscopic technique using an SH2-containing phosphotyrosine reporter to monitor tyrosine phosphorylation in focal adhesions in live cells is described.
Iyer
Vidhya V
VV
Department of Cell and Developmental Biology, University of North Carolina-Chapel Hill, Chapel Hill, NC, USA.
Ballestrem
Christoph
C
Kirchner
Joachim
J
Geiger
Benjamin
B
Schaller
Michael D
MD
D016475:3T3 Cells; D000818:Animals; D002448:Cell Adhesion / Q000502:physiology*; D002642:Chick Embryo; D003598:Cytoskeletal Proteins / Q000378:metabolism; D005109:Extracellular Matrix / Q000502:physiology / Q000648:ultrastructure; D007150:Immunohistochemistry / Q000379:methods; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*
D003598:Cytoskeletal Proteins; D051419:Paxillin; D010750:Phosphoproteins; C496062:Pxn protein, mouse; D019000:Phosphotyrosine