Probing endogenous mTORC2 activity using LocaTOR. (A) LocaTOR2 phosphorylation in subcellular membrane compartments is mTOR dependent. HEK293T cells were cotransfected with LocaTOR2 and the corresponding recruiter constructs, serum starved overnight, and incubated with 250 nM Torin1 before (pre) or after (post) a 40-min treatment with 250 nM AP21967. endo, endogenously expressed Akt. (B) LocaTOR2 phosphorylation is restricted to the recruiting compartments. HEK293T cells were cotransfected with FRB:Akt2-citrine (bottom, gray) and the corresponding recruiter constructs (top) for 18 h and serum starved overnight. After 40 min of incubation with 250 nM AP21967, the cells were fixed, permeabilized, and immunostained with Akt^pS473 phosphospecific antibody (bottom, red). Bars, 5 µm. (C) Quantification of the results shown in B. The images of mCherry-tagged recruiter constructs were segmented to create binary masks, in which mean fluorescence intensity in the phosphospecific antibody channel was determined and normalized on a cell-by-cell basis by the mean intensity of FRB:Akt2-citrine fluorescence. Data are presented as box plots (red, median; whiskers, 5th and 95th percentiles). Numbers in parentheses indicate the number of cells analyzed from two to three independent experiments. Mann-Whitney U test: ***, P < 0.001. EE, early endosome; LE, late endosome; PM, plasma membrane; mito, outer mitochondrial membrane; N, nontransfected control.