Figure 1.
Figure 1. LocaTOR2 reports local mTORC2 activity. (A) Scheme of the compartment-specific reporter. Cytosolic FRB:Akt2 can be recruited to the plasma membrane using AP21967 rapalog, and FRB:Akt2 phosphorylation can be monitored using specific antibodies. (B and C) AP21967 induces translocation of FRB:Akt2-citrine to subcellular membrane compartments. Recruitment half time was determined by calculating Pearson’s coefficient of the recruiter channel (mCherry, red in second and third row; B) and the citrine channel (green; B). Bars, 10 µm. Data are presented as means ± SD; the number of cells analyzed is shown in parentheses (C). (D) FRB:Akt2 phosphorylation is sensitive to Torin1, but not to rapamycin. HEK293T cells were transfected with LocaTOR2 for 18 h, treated for 40 min with 250 nM Torin1, 275 nM rapamycin, or the vehicle, and lysed, and the lysates were analyzed using PAGE/WB. endo, endogenously expressed Akt.

LocaTOR2 reports local mTORC2 activity. (A) Scheme of the compartment-specific reporter. Cytosolic FRB:Akt2 can be recruited to the plasma membrane using AP21967 rapalog, and FRB:Akt2 phosphorylation can be monitored using specific antibodies. (B and C) AP21967 induces translocation of FRB:Akt2-citrine to subcellular membrane compartments. Recruitment half time was determined by calculating Pearson’s coefficient of the recruiter channel (mCherry, red in second and third row; B) and the citrine channel (green; B). Bars, 10 µm. Data are presented as means ± SD; the number of cells analyzed is shown in parentheses (C). (D) FRB:Akt2 phosphorylation is sensitive to Torin1, but not to rapamycin. HEK293T cells were transfected with LocaTOR2 for 18 h, treated for 40 min with 250 nM Torin1, 275 nM rapamycin, or the vehicle, and lysed, and the lysates were analyzed using PAGE/WB. endo, endogenously expressed Akt.

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