Cell-based assays to probe the ERK MAP kinase pathway in endothelial cells.

To understand signaling pathways in mammalian cells, cell-based assays are relatively new and extremely powerful tools. We have developed a battery of phenotypic assays to study signaling; two of them are described in detail in this chapter. A subset of these assays monitors mitogen-activated protein (MAP) kinase pathways. MAP kinases are principal regulators of fundamental processes in mammalian cells, including growth, cell division, differentiation, stress responses, and neoplastic transformation. Here we describe two cell-based assays querying the function of ERK (extracellular signal regulated kinase), one of the three principal MAP kinases in mammalian cells. We selected human umbilical vein endothelial cells (HUVECs), a primary cell type, because they show a very dynamic response to various activators. Both assays are phenotypic assays and use well-established phosphorylation-specific primary antibodies to study activation. Fluorochrome-coupled secondary antibodies were used to label phosphorylated target proteins; images were captured with the INCell Analyzer 3000 and analyzed with the INCell Analyzer 3000 software. The first of these two assays monitors phosphorylation of ERK1/2, while the second assay monitors activation of the transcription factor CREB (cAMP response element-binding protein). The assays described in this chapter cover major checkpoints of the ERK signaling pathway: (1) MAP kinase activation and (2) subsequent transcription factor activation. Both assays exhibit robust performance and can easily be used for high-throughput screening.