Figure 2.
UT2 Interacts with mTORC2/RICTOR and Regulates Their Activities in Primary Hematopoietic Cells
(A) HEK293T cells transfected with vectors encoding either Flag-UT2 full length or Flag-UT2 ΔC mutant were lysed and subjected to IP using an antibody directed against FLAG and IgG, respectively. The resulting precipitates and corresponding whole-cell lysates (WCLs) were subjected to western blot analysis using the indicated antibodies (n = 3 experiments).
(B) RICTOR or UT2 was immunoprecipitated from WT and conditional homozygous deletion of Rictor BM cells and subjected to western blot analysis using the indicated antibodies (n = 6 mice pool per each genotype).
(C) In vitro-translated FLAG-UT2 full length (left) or FLAG-UT2 ΔC mutant (right) proteins were subjected to IP using the indicated antibodies in the presence of in vitro-translated myc-RICTOR protein (n = 2–3 experiments).
(D) WT and conditional homozygous deletion of Rictor BM cells expressing shRNA constructs against control or UT2 were subjected to western blot analysis using the indicated antibodies (n = 3 mice pool per each genotype).
(E) Endogenous RICTOR was immunoprecipitated from control and HEK293T cells transfected with vectors encoding FLAG-UT2 full length and subjected to in vitro kinase assays using recombinant inactive AKT as the substrate and pAKTS473 as the readout. An immunoblot for the levels of RICTOR, mTOR, and mLST8 in each immunoprecipitate is shown (n = 3 experiments).
See also Figure S2.