Fig 2.
UCH-L1 depletion leads to increased mTORC1 activity. (A) KMS-28doxsh cells were incubated with or without doxycycline (Dox) for 5 days and were then analyzed by immunoblotting using the indicated antibodies. (B) Sizes (FSC-H) of KMS-28doxsh cells from panel A as measured by flow cytometry. (C) Brain tissue was harvested from age-matched mice of the indicated genotypes, followed by immunoblotting using the indicated antibodies. (D) HeLa cells with or without stable expression of UCH-L1 were grown under standard conditions and analyzed by confocal immunofluorescence microscopy using the indicated antibodies. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole). (E) HeLa cells with or without stable expression of UCH-L1 were starved of amino acids (−AA) for 50 min and restimulated with amino acids as detailed in Materials and Methods. The cells were fixed and stained for mTOR as indicated. Bars = 5 μm.