4 Notes
Full personal protective equipment including gloves has to be worn
for the whole procedure to reduce contamination throughout the
preparation. Sterile conditions are required particularly for the cell
culture.
Acknowledgments
This work was supported by a BBSRC iCASE PhD studentship
(BB/L016052/1) to AA. We thank STFC for funding access to the
Central Laser Facility. This work also acknowledges Professor Raymond Owens (PP-UK) for providing support and plasmid vectors
for the cloning work.
References
1. Beretta L, Gingras AC, Svitkin YV et al (1996)
Rapamycin blocks the phosphorylation of
4E-BP1 and inhibits cap-dependent initiation
of translation. EMBO J 15(3):658–664
2. Brunn GJ, Hudson CC, Sekulic ´ A et al (1997)
Phosphorylation of the translational repressor
PHAS-I by the mammalian target of rapamycin. Science 277(5322):99–101
Table 6
Three-color FRET-FLIM results for multiple mTORC1 interactions
mTORC1 proteins
Lifetime (ns)
Comment
S6K1-mTurq2
4.3 Æ 0.1
Control lifetime
YFP-Rheb
2.8 Æ 0.1
Control lifetime
YFP-mTOR
~2.8 Æ 0.1
Control lifetime
mCherry-raptor
N/A
τ not measured
S6K1-mTurq2 + mCherry-raptor
3.3 Æ 0.1
Interaction
YFP-Rheb + mCherry-raptor
2.8 Æ 0.1
No interaction
YFP-mTOR + mCherry-raptor
2.5 Æ 0.1
Interaction
S6K1-mTurq2 + YFP-Rheb +
mCherry-raptor
3.2 (ch 1) + 2.8
(ch 2) Æ 0.1
Interaction between S6K1 and raptor,
but no interaction between S6K1
and Rheb or raptor and Rheb.
No direct three-way interaction
between mTOR, raptor, and S6K1
S6K1-mTurq2 +
YFP-mTOR + mCherry-raptor
3.1 (ch1) + 2.5
(ch 2) Æ 0.1
Strong interaction between S6K1 and
raptor strong interaction between
raptor and mTOR. Possible slight
direct three-way interaction between
mTOR, raptor, and S6K1
Multicolor FRET-FLIM Microscopy to Analyze Multiprotein Interactions in. . .
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