ABSTRACT
A number of technical approaches can be used to analyze the intracellular signaling pathways that regulate integrin activity. One approach is the use of inhibitors that block the activity of specific signaling pathways. Another approach for disrupting signaling pathways is to overexpress wild-type or mutant forms of kinases and adapter proteins in cells, and determine the effects of the expression of these proteins on the functional response of interest. This chapter provides a detailed description of the assay and the subsequent data analysis, using CD2+ HL–60 cells, a GFP fusion protein expressing a dominant-negative form of the p85 subunit of PI 3-K, and adhesion to fibronectin as an example. The chapter utilizes flow cytometry to analyze the adhesive behavior of a phenotypically distinct subset of cells without having to physically separate this subset before performing the adhesion assay.
