Measuring phosphorylated Akt and other phosphoinositide 3-kinase-regulated phosphoproteins in primary lymphocytes

Phosphoinositide 3-kinase (PI3K) is a lipid kinase whose activation is crucial for many biological functions in multiple cell types. One research area of particular interest for basic biologists and drug developers is PI3K signaling in lymphocytes. Inhibitor studies and PI3K mutants have demonstrated that PI3K is required for development, activation, proliferation, differentiation, and survival of B lymphocytes, as well as optimal activation and proliferation of T lymphocytes. As the actual products of PI3K can be difficult to measure, the field has often adopted the practice of examining the activation of downstream effectors of PI3K, with the most common readout being phosphorylation of Akt. This chapter discusses key pathways influenced by PI3K signaling and the advantages and caveats of using activation of these pathways as indicators of PI3K activity. In addition, we provide traditional immunoblotting methods of assaying PI3K-dependent pathway activation, as well as more recent flow cytometry-based approaches (termed "phosflow"). Although we describe assays optimized for B lymphocytes, these methods are easily adapted to T lymphocytes and other leukocyte cell types.