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. 2019 Mar 19;14(3):e0213701.
doi: 10.1371/journal.pone.0213701. eCollection 2019.

Expression of Concern: Akt Regulates Drug-Induced Cell Death through Bcl-w Downregulation

PLOS ONE Editors

Expression of Concern: Akt Regulates Drug-Induced Cell Death through Bcl-w Downregulation

PLOS ONE Editors. PLoS One. .
No abstract available

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Figures

Fig 2
Fig 2. Akt activity regulates Bcl-w expression.
(A) HeLa cells were transfected with 2 μg of HA-Akt wt, Akt D+, or HA-Akt D− cDNA and 2 μg Flag-Bcl-w for 48 hrs. Protein extracts were immunoprecipitated with an anti-HA monoclonal antibody. Immunoprecipitates were resolved on 12% SDS-PAGE and transferred to Hybond-C nitrocellulose. Membranes were incubated with anti-Flag antibody (0.2 μg/ml). 50 μg of total sample extracts were also analyzed by western blot using the indicated antibodies. Loading control was obtained using anti-β actin antibody. (B) HeLa cells were transfected with 4 μg of HA-Akt wt, HA-Akt D+, or HA-Akt D− cDNA for 48 hrs. Protein extracts were blotted with anti-Bcl-w antibody in order to detect endogenous levels of Bcl-w. Loading control was obtained with anti-β actin antibody. (C) Cells were transfected with 100 nM of siAkt-RNA for 48 hrs. Cellular proteins were solubilized and analyzed by western blot using the indicated antibodies. (D) HeLa cells were treated with 10, 20 or 40 μM of LY294002 for 24 hrs. Protein extracts were analyzed by western blot using the indicated antibodies. Loading control was obtained using anti-β-actin antibody. (E) Bcl-w HeLa cells were treated with 10 μM of MG-132 for 8 hrs. 40 μg of protein extracts were analyzed by western blot with anti-Bcl-w antibodies. Loading control was obtained using anti-β actin antibody.
Fig 4
Fig 4. Akt phosphorylates Bcl-w in vitro and in vivo.
(A) HeLa cells were transfected with 2 μg of DNA of Flag Bcl-w, solubilized, and 1 mg of protein extract was immunoprecipitated with an anti-M2 Flag antibody. Immunoprecipitates were incubated with recombinant constitutive active Akt (rAkt), and in vitro kinase assay was conducted as described in the methods. Samples were loaded onto 2.5% SDS-PAGE and analyzed by autoradiography. As positive control we used Histone2B (H2B). (B) HeLa Bcl-w stable expressing clones were serum starved for 18 hrs and then stimulated with 100 nM insulin or with 20% serum for 15 min as indicated. Cells were solubilized and immunoprecipitated with an anti-M2 Flag antibody. Immunoprecipitates were loaded onto SDS-PAGE and blotted with an anti-phospho Akt substrate antibody that recognizes all the phosphorylated Akt substrates. Total extracts were analyzed by western blot using the indicated antibodies. (C) HeLa cells were transfected with 2 μg of HA-GSK3β and either 2 μg of pcDNA3 empty vector (lane 2) or 2 μg of Flag-Bcl-w (lane 3) for 48 hrs. Cells used for the samples in lane 1 were untransfected controls (C-). Cells were stimulated with 100 nM insulin for 15 min, solubilized, immunoprecipitated using an anti-HA antibody, and analyzed by western blot using an anti-phospho-Gsk3β antibody (top panel). Total extracts were analyzed by western blot using anti-Flag, anti-HA, and anti-β-actin antibodies (panels 2–4, respectively). Bcl-w overexpression does not affect Akt activity. Vertical lines in Fig 4A and 4C indicate where fragments of the original blot images were spliced to remove or rearrange lanes. The raw uncropped blots are provided in S4 File. Note that the Flag-Bcl-w and HA-GSK3β data in the second and third panels of Fig 4C were obtained using independent blots for which the corresponding β-Actin control blots are provided in S4 File. The β-Actin panel shown in the figure is for the anti-HA blot, but the authors clarified that the same protein extracts were used for both blots.
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References

    1. Garofalo M, Quintavalle C, Zanca C, De Rienzo A, Romano G, Acunzo M, et al. (2008) Akt Regulates Drug-Induced Cell Death through Bcl-w Downregulation. PLoS ONE 3(12): e4070 10.1371/journal.pone.0004070 - DOI - PMC - PubMed
    1. Quintavalle C, Incoronato M, Puca L, Acunzo M, Zanca C, Romano G et al. (2010) c-FLIPL enhances anti-apoptotic Akt functions by modulation of Gsk3β activity. Cell Death and Differentiation 17, 1908–1916. https://www.nature.com/articles/cdd201065 - PubMed
    1. (2017) Retraction to: Cell Death and Differentiation 2010; 17: 1908–1916. 10.1038/cdd.2010.65 Cell Death and Differentiation 24, 1134. https://www.nature.com/articles/cdd20177 - DOI - PubMed

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