Fig. 4: Ser 9 phosphorylation by AKT1 attenuates mitochondrial translocation of ME2fl.
From: AKT1 phosphorylation of cytoplasmic ME2 induces a metabolic switch to glycolysis for tumorigenesis
a Confocal imaging of U2OS cells expressing ME2fl-3’Flag or ME2fl truncates (ME2fl∆1-9-3’Flag and ME2fl∆1-18-3’Flag). ME2fl was immune-stained with an anti-Flag antibody, mitochondria were stained with MitoTracker and DNA was stained with DAPI. Scale bars,10 μm. b Fractionation was performed on HEK293T cells expressing ME2fl-3’Flag or ME2fl truncates (ME2fl∆1-9-3’Flag and ME2fl∆1-18-3’Flag). The expression of ME2fl and its truncations in whole-cell lysates, cytoplasmic and mitochondrial fractions were analyzed by western blotting using an anti-Flag antibody. β-tubulin and COXIV served as loading controls as well as cytosolic and mitochondrial markers, respectively. c Immunofluorescence analysis of ME2 in U2OS cells expressing ME2fl-3’Flag, ME2flS9A-3’Flag and ME2flS9D-3’Flag using an anti-Flag antibody. Mitochondria were stained with MitoTracker and DNA was stained with DAPI. Scale bars,10 μm. d HEK293T cells were transfected with ME2fl-3’Flag (WT) and ME2flS9A-3’Flag or ME2flS9D-3’Flag, followed by fractionation. The fractionations were analyzed by western blot using indicated antibodies. β-tubulin and COXIV served as loading controls as well as cytosolic and mitochondrial markers, respectively. e Confocal imaging of U2OS cells expressing 3’GFP-tagged 1-18AA peptide (P1-18/WT-3’GFP) and mutant 1-18AA peptides (P1-18/S9A-3’GFP and P1-18/S9D-3’GFP). Mitochondria were stained with MitoTracker and DNA was stained with DAPI. Scale bars, 10 μm. f HEK293T cells expressing 3’GFP-tagged 1-18AA peptide (P1-18/WT-3’GFP) and mutant 1-18AA peptides (P1-18/S9A-3’GFP and P1-18/S9D-3’GFP) were subcellularly fractionated and analyzed by immunoblotting with an anti-Flag antibody. β-tubulin and COXIV served as loading controls as well as cytosolic and mitochondrial markers, respectively. g Confocal imaging of U2OS cells expressing ME2fl-3’Flag, ME2flS9A-3’Flag or ME2flS9D-3’Flag together with HA-AKT1 or HA vector control as indicated. ME2fl and AKT1 was immune-stained with the anti-Flag and anti-HA antibodies respectively, mitochondria were stained with MitoTracker and DNA was stained with DAPI. Scale bars, 10 μm. All data are representative of three independent experiments.
