Fig. 3.

Skp2 and AMPK are crucial for stress-induced Akt ubiquitination and activation. a HEK293 cells in normoxia and hypoxia were subject to immunoprecipitation with Ubiquitin antibody, followed by immunoblotting. Input is 10% of whole cell lysate. b In vivo ubiquitination assay of Akt from 293T cells transfected with WT, K63R and K48R His-Ub in response of 1% O₂. Input is 10% of whole cell lysate. c Control (shLuc) and AMPKα1 knockdown MDA-MB-231 cells in normoxia and hypoxia were subjected to immunoprecipitation with K63-specific Ubiquitin antibody, followed by immunoblotting. Input is 10% of whole cell lysate. d–f In vivo ubiquitination assay of Akt from WT, AMPKα^-/- MEFs restored with vector and WT AMPKα together with transfection of HA-Akt and His-Ub in response of hypoxia (1% O₂), glucose deprivation and H₂O₂ for indicated time. Input is 10% of whole cell lysate. g WT and Skp2^-/- MEFs challenged with 1% O₂ for 8 h were subjected to immunoprecipitation with K63-specific Ubiquitin antibody, followed by immunoblotting. h Immunoblotting of WT and Skp2^-/- MEFs that were challenged with 1% O₂ for 4 and 8 h. i Immunoblotting of control (shLuc) and Skp2 knockdown (#1) MDA-MB-231 cells were serum starved and treated with H₂O₂ (100 μM) for indicated time