Quantitative Monitoring Spatiotemporal Activation of Ras and PKD1 Using Confocal Fluorescent Microscopy

Receptor activation upon ligand binding induces activation of multiple signaling pathways. To fully understand how these signaling pathways coordinate, it is essential to determine the dynamic nature of the spatiotemporal activation profile of signaling components at the level of single living cells. Here, we outline a detailed methodology for visualizing and quantitatively measuring the spatiotemporal activation of Ras and PKD1 by applying advanced fluorescence imaging techniques, including multichannel, simultaneous imaging and Förster resonance energy transfer (FRET).