FIGURE 5.

NHE3 and NHERF3 directly associate as determined by fluorescence resonance energy transfer and this association decreases in response to elevated calcium. PS120 cells were prepared as described above and NHE3 and NHERF3 protein expression detected using secondary antibodies conjugated to Cy3 (NHE3) or Cy5 (NHERF3). A, NHE3 and NHERF3 directly interact in PS120/NE3V/NHERF3 cells as determined by acceptor photobleaching FRET. Using confocal microscopy, initial fluorescent images of NHE3 and NHERF3 localization were captured before photobleaching (a, a′, c, and c′). Cy5-labeled NHERF3 signal was bleached with continuous laser excitation until Cy5 fluorescence was completely eliminated (d and d′). NHE3 Cy3 (donor) images (b and b′) were captured after Cy5 photobleaching and analyzed for changes in fluorescent intensity using the MetaMorph image analysis software. B, the results of the current study demonstrated that NHE3 and NHERF3 exhibit FRET under basal conditions and the total percent of energy transfer was significantly (p < 0.01) decreased in PS120/NHE3V/NHERF3 cells treated for 15 min with 4-bromo-A23187. 50–100 regions of interest were analyzed and data summarized in B. *, p in comparison to control.