Figure 5.
Figure 5. Akt membrane recruitment is sufficient for Ser473 phosphorylation by mTORC2. (A and B) Model showing reversible recruitment strategy (A). HeLa cells were cotransfected with FKBP:mCherry-Akt2K14A and TagBFP-2×DHFR-CAAX constructs for 18 h and then treated with 1 µM SLF'-TMP for 12 min, after which 10 µM TMP was added (mean fluorescence intensity in the cytosolic region of interest). Data are presented as means ± SD; seven cells. PM, plasma membrane. (C) Akt membrane recruitment is necessary and sufficient for pSer474 phosphorylation. HeLa cells were cotransfected with FKBP:mCherry-Akt2K14A and TagBFP-2×DHFR-CAAX constructs for 18 h and then treated with 1 µM SLF'-TMP for 30 min, after which 10 µM TMP was added for the time indicated. The cells were lysed, and pSer473 phosphorylation was determined using PAGE/WBs. Shown is one representative out of three experiments. endo, endogenously expressed Akt.

Akt membrane recruitment is sufficient for Ser473 phosphorylation by mTORC2. (A and B) Model showing reversible recruitment strategy (A). HeLa cells were cotransfected with FKBP:mCherry-Akt2K14A and TagBFP-2×DHFR-CAAX constructs for 18 h and then treated with 1 µM SLF'-TMP for 12 min, after which 10 µM TMP was added (mean fluorescence intensity in the cytosolic region of interest). Data are presented as means ± SD; seven cells. PM, plasma membrane. (C) Akt membrane recruitment is necessary and sufficient for pSer474 phosphorylation. HeLa cells were cotransfected with FKBP:mCherry-Akt2K14A and TagBFP-2×DHFR-CAAX constructs for 18 h and then treated with 1 µM SLF'-TMP for 30 min, after which 10 µM TMP was added for the time indicated. The cells were lysed, and pSer473 phosphorylation was determined using PAGE/WBs. Shown is one representative out of three experiments. endo, endogenously expressed Akt.

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