Figure 2: The cytoplasmic domain of mIgG associates with the PM in quiescent B cells.

(a) Schematic representation showing the FRET system that was used to detect the interaction between mTFP (FRET donor) fused to C terminal of cytoplasmic tail and R18 dye (FRET acceptor) stained on the PM. FRET efficiency was calculated as detailed in the Method section. (b,c) Dequencing FRET to measure the FRET efficiency between mTFP and R18 in KIR-3aa, KIR-25aa and KIR-mIgG-tail expressing A20 B cells. FRET efficiency was calculated as detailed in the Method section. Representative confocal images were shown for each group (b). FRET efficiency was measured and plotted (c). Scale bar is 1.6 μm. (d) Similar as in a shown is a schematic FRET system to measure the interaction of the mIgG-tail with the inner leaflet of the PM in the context of IgG-BCR complex. Used as a control is mIgM-tail in the context of IgM-BCR complex. (e,f) Representative confocal images of dequenching FRET in IgM-BCR or IgG-BCR expressing J558L cells (e). FRET efficiency was calculated and compared in the context of IgM-BCR versus IgG-BCR (f). Scale bar, 1.6 μm. For all the FRET efficiency measurements, each dot represents one cell analysed in three independent experiments. Bars indicate mean±s.d. Two-tailed t-tests were used for the statistical comparisons. ***P<0.001; **P<0.01; NS, not significant.