Donahue Amber C, Kharas Michael G, Fruman David A
Department of Molecular Biology and Biochemistry and Center for Immunology, University of California-Irvine, Irvine, California, USA.
Methods Enzymol. 2007;434:131-54. doi: 10.1016/S0076-6879(07)34008-1.
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.
磷脂酰肌醇3激酶（PI3K）是一种脂质激酶，其激活对于多种细胞类型的许多生物学功能至关重要。基础生物学家和药物研发人员特别感兴趣的一个研究领域是淋巴细胞中的PI3K信号传导。抑制剂研究和PI3K突变体表明，PI3K是B淋巴细胞发育、激活、增殖、分化和存活以及T淋巴细胞最佳激活和增殖所必需的。由于PI3K的实际产物可能难以测量，该领域通常采用检测PI3K下游效应器激活的做法，最常见的读数是Akt的磷酸化。本章讨论受PI3K信号传导影响的关键途径以及将这些途径的激活用作PI3K活性指标的优点和注意事项。此外，我们提供了检测PI3K依赖性途径激活的传统免疫印迹方法，以及更新的基于流式细胞术的方法（称为“磷酸化流式细胞术”）。虽然我们描述了针对B淋巴细胞优化的检测方法，但这些方法很容易适用于T淋巴细胞和其他白细胞类型。
