Figure 11

EGFP-mTOR direct interaction with DsRed-raptor in HEK293 cells and lack of effect of rapamycin. (A, B) LEFT PANELS: Confocal 488 nm excitation images of EGFP-mTOR (co-expressed with DsRed-raptor, after 48 h of transfection) and 543 nm excitation images of DsRed-raptor, without (A) and with (B) rapamycin (100 nM) treatment for 24 h. (A, B) MIDDLE PANELS: Lifetime images of the co-transfected cells (colour coding for the lifetimes is shown in the RIGHT PANELS). (A, B) RIGHT PANELS The lifetime distributions taken for the the region taken here is within the area of the thin red line (not the entire area as previous Figures) as shown in the MIDDLE PANELS. The lifetime value from the data in (A) RIGHT PANEL for the EGFP (attached to mTOR) is reduced due to quenching by DsRed (attached to raptor) from ~2450 ± 100 ps for EGFP alone (see Figure 9) to 2300 ± 100 ps (n=3) here, thus showing a direct interaction. The data in (B) RIGHT PANEL for the lifetime of the EGFP (attached to mTOR) for the cell demarked by the thin red line (the cell below being omitted as it is not expressing DsRed-raptor, see LEFT PANEL), after treatment with rapamycin there was no effect on the lifetime centre (2300 ps ± 100 ps). The data collection time for lifetime images was optimally 3 accumulations of 30 sec. with an image dimension of 128×128 pixels, bar 8 μm.