Figure 10

DsRed-Rheb direct interaction with EGFP-mTOR 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-Rheb, after 48 h of transfection) and 543 nm excitation images of DsRed-Rheb, 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 entire area (white border) 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 by quenching by DsRed (attached to Rheb) from ~2450 ± 100 ps for EGFP alone (see Figure 9) to 2200 ± 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), after treatment with rapamycin there was no relief of the quenching indicating it does not impact on the direct interaction. 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.