Figure 1. Identification of GATOR2 components as Sestrin2-binding partners.

(A) Sestrin2 inhibits mTORC1 in AMPK-null cells. WT and AMPK-null (Ampkα₁^−/−/Ampkα₂^−/−) MEFs were infected with adenoviruses expressing GFP or Sestrin2 and analyzed through immunoblotting (IB). (B) Mass spectrometry analysis of Sestrin2-tandem affinity purification (TAP) products identified GATOR2 components as proteins that show the strongest physical interaction with Sestrin2. Sestrin2 TAP products from MCF10A cells were analyzed by MS-MS. The number of peptide hits for Sestrin2 and its six strongest interacting partners, among which five are GATOR2 components (shaded in grey), are shown. (C) GATOR2 as a complex shows strong physical association with Sestrin2 in HEK293 cells. Sestrin2 was co-transfected with all GATOR2 components as indicated. Sestrin2 was immunoprecipitated (IPed) with Flag antibody. Input (whole cell lysate, WCL) and IP complex were analyzed by IB. (D) Endogenous Sestrin2 interacts with endogenous GATOR2 proteins in livers from obese mice. Sestrin2 and its interacting proteins in liver lysates of 4-month-old Lep^ob/ob/Sesn2^+/− and Lep^ob/ob/Sesn2^−/− mice were IPed with Sestrin2 antibody. Input (WCL) and IP complex were analyzed by IB with indicated antibodies against endogenous proteins. (E) Endogenous Sestrin2 interacts with endogenous GATOR2 proteins in mouse embryonic fibroblast (MEF) cells treated with 100 μM etoposide, a DNA damage inducer that increases Sestrin2 expression, for 16 hrs. Sestrin2 and its interacting proteins were IPed with Sestrin2 antibody or control immunoglobulin (IgG). Input (WCL) and IP complex were analyzed by IB with indicated antibodies against endogenous proteins. Cropped gel images are used in this figure and the gels were run under the same experimental conditions.