329063.fig.004a
(a)
329063.fig.004b
(b)
Figure 4: Kinase-inactive PKCε colocalizes with RhoA at the cell membrane. (a) Colocalization of kinase-inactive PKCε and RhoA. HEK293 cells were cotransfected with mCherry-PKCε/K437R and eGFP-RhoA. Fluorescence images were captured prior to and 15 minutes after PMA (100 nM) stimulation. (b) Stoichiometric FRET analysis of kinase-inactive PKCε and RhoA interaction. HEK293 cells were cotransfected with mCherry-PKCε/K437R 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/K437R (acceptor image, IA), eGFP-RhoA (donor image, ID), and the FRET interaction between mCherry-PKCε/K437R 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.