Figure 3.

Detection of the DLG1-Tax interaction by fluorescence resonance energy transfer (FRET) microscopy. (A) Acceptor photobleaching FRET experiments. HEK293 epithelial cells transiently transfected with pmTurq2-DLG1 and pseyfp2-Tax were submitted to the acceptor photobleaching FRET protocol described in Experimental Procedures. Changes in the intensity of mTurq2-DLG1 emission can be appreciated (donor, left panel) in the region of interest (ROI) under study before and after photobleaching of the seyfp2-Tax emission (acceptor, right panel). The mTurq2-DLG1 fluorescence emission is shown in a pseudocolored table which allows for a better visualization of the changes in fluorescence intensity. The vertical scale on the left displays low to high intensities from bottom-up. The representative ROI under study is delimited in a white square and is shown magnified in the inset. All scale bars are 10 µm; (B) Quantification of the FRET efficiency in photobleaching experiments. The mTurq2-DLG1 mean fluorescence intensity in the ROI under study was determined before and after photobleaching of seyfp2-Tax emission. These two values were introduced into the formula described in Experimental Procedures and the FRET efficiency (%) for that particular ROI was obtained (n = 15, left side, blue bar). To ascertain the background FRET efficiency, this protocol was simultaneously applied over a random non photobleached ROI inside the same cell (n = 15, left side, green bar). On the other hand, as negative biological controls, the whole procedure was also carried out in cells expressing mTurq2-DLG1 and seyfp2-TaxMUT (n = 8, middle blue and green bars) or control mTurq2 along with seyfp2-Tax (n = 8, right side, blue and green bars). In each condition, FRET efficiency % are shown as Mean ± SD. Conditions tested for statistical significance are shown with horizontal lines and assessed by a One-tailed Student t-test. * p < 0.05 was considered to be significant.