Figure 3: Stoichiometric FRET analysis of PKCε-RhoA interaction in live cells. HEK293 cells were cotransfected with mCherry-PKCε and eGFP-RhoA. Fluorescence images were captured prior to and every 15 seconds after PMA (100 nM) stimulation for 12.5 minutes and then subjected to quantitative FRET analysis. The images presented at each time point represent mCherry-PKCε (acceptor image, IA), eGFP-RhoA (donor image, ID), and the FRET interaction between mCherry-PKCε and eGFP-RhoA (ED). The color bars at the end of the panels indicate the scaling of the ED images with warmer colors representing higher values. Time course of normalized ED for the cell membrane and cytoplasm is presented. ED was normalized to the ED of the cytoplasm at the 0 minute time point. Cell membrane is defined as ±1 μm from the cell membrane border. Cytoplasm is defined as all intracellular space, including the nucleus, 1 μm from the cell membrane border.