Figure 5.

FRET analysis of the interaction between ORP3, VAP-A/B, and Rab7 in NEI. A–F, potential interactions between ORP3, VAP proteins, and Rab7 were evaluated by FRET using the acceptor photobleaching method. When appropriate, FEMX-I cells expressing VAP-A–GFP and/or Rab7–RFP were immunolabeled for ORP3 or VAP-B followed by secondary antibody coupled to appropriate fluorophore (TRITC or FITC). Intensity profiles and FRET efficiencies of each pair in NEI (A–D) or cytoplasm (E and F) are presented. The means ± S.D. are shown (n = 3). At least five cells were evaluated per experiment, and all of them are plotted. Acceptor photobleaching with a 561-nm laser line starts at time 15 s. FRET efficiencies were measured in a given ROI in bleached and unbleached areas. Corresponding micrographs with ROI are shown in Fig. S7.