Fig. 6.

Effect of Cer, GlcCer, SPM, and Fg on formation of functional caveolae in MBECs. Formation of caveolae was determined by Förster resonance energy transfer (FRET) and total internal reflection fluorescence (TIRF) microscopy. Cells were transfected with green fluorescence protein (GFP)-labeled plasmalemmal vesicle-associated protein-1 (PV-1) and/or mCherry-labeled Na⁺-K⁺-ATPase, and live cells were imaged as described in methods. A, B, and D: representative images from 3 individual experiments for epifluorescence (A), FRET (B), and TIRF (D). Merged images in B are in pseudocolor (gated to mCherry acceptor levels). Color scale shows reference spectrum: blue indicates no association (FRET and TIRF), and red indicates association (FRET and TIRF). C: data for 3-channel FRET efficiency after photobleaching from 3 individual experiments. In each experiment, FRET efficiency from 30–50 cells was calculated, averaged, and considered as 1 experimental value. E and F: expression of Na⁺-K⁺-ATPase and caveolin-1 in the plasma membrane was determined by TIRF, and the number of caveolae was counted as individual GFP (E) and mCherry (F) particles using ImageJ software. In each experiment, values from 30–50 cells were averaged and considered as 1 experimental value. Values are means ± SE; n = 3. *P < 0.05 vs. control.