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. 2009 May 22;284(21):13987–14000. doi: 10.1074/jbc.M901758200

FIGURE 2.

FIGURE 2.

MDM2 interacts with FOXO1 and FOXO3A in vivo and in vitro. A, colocalization of ectopic MDM2 and FLAG-FOXO1 in p53 and MDM2 double null MEFs. The fibroblasts were transfected with 0.5 μg of FLAG-FOXO1 and 0.5 μg of MDM2 and fixed. The colocalization was determined by immunofluorescence staining with anti-MDM2 and M2 anti-FLAG antibodies and visualized by deconvolution imaging. B, colocalization of endogenous MDM2 and FOXO3A. H1299 cells were fixed and subjected to immunofluorescence staining with DAPI, anti-MDM2, and anti-FOXO3A, as indicated. The colocalization was visualized by confocal imaging. C, interaction between ectopic FOXO1 and MDM2. H1299 cells were transfected with 2 μg of HA-FOXO1, 2 μg of MDM2, or both. 24 h posttransfection, cellular extracts were prepared and subjected to co-precipitations (IP) and immunoblotting (IB) with the indicated antibodies. D, interaction of ectopic FOXO3A and MDM2. H1299 cells were transfected with 2 μg of MDM2 and 2 μg of GFP or GFP-FOXO3A. 24 h posttransfection, cellular extracts were prepared and subjected to co-precipitations and immunoblotting with the indicated antibodies. E, interaction of endogenous MDM2 and FOXO1 in HI299/V138 cells. HI299/V138 cells were cultured at a permissive temperature (32 °C) overnight and treated with vehicle (DMSO) or MG132 for 6 h. Cellular extracts were subjected to co-immunoprecipitations, followed by immunoblotting with the indicated antibodies. M, anti-MDM2; HA, anti-HA (control). F, interaction of endogenous MDM2 and FOXO3A. HEK293T cells in a 100-mm dish were treated with DMSO or MG132 for 6 h. Then cellular extracts were prepared and subjected to co-immunoprecipitations, followed by immunoblotting with the indicated antibodies. M, anti-MDM2; HA, anti-HA (control). G, in vitro interaction between FOXO1 and MDM2. FOXO1 produced in in vitro transcription-coupled translation reactions was incubated with either GST or GST-MDM2 fusion proteins. Pull-down assays were performed with magnetic glutathione beads, followed by immunoblotting with anti-FOXO1 antibody. The amount of GST and GST-MDM2 proteins used in the pull-down assays was visualized by Coomassie Blue staining after SDS-PAGE (bottom).