Journal: Cell death and differentiation

Article Title: Hdm2 is an Ubiquitin Ligase of Ku70 -Akt Promotes Cell Survival by Inhibiting Hdm2-dependent Ku70 Destabilization

doi: 10.1038/cdd.2009.6

Figure Lengend Snippet: a and b, Akt did not show cytoprotection in Ku70 siRNA treated cells. HeLa cells were plated onto six-well plates at 1.2×10 5 cells per well and transfected with the Ku70 siRNA (SiKu70) or scrambled control RNA (Scr) as described . Twenty-four h after transfection, cells were infected with adenovirus expressing Myc-Myr-Akt (constitutively active) andincubated additional 24 h. Then, cells were treated with 25 uM Etoposide (Etopo) ( a ) or 200 nM staurosporin (STS) ( b ) for 16 h, and apoptosis induction was determined by Hoechst-dye nuclear staining. Data represent averages (± S.E.) (300 cells per sample were counted) of triplicated experiments. c, Ku70 siRNA successfully knocked down Ku70 in HeLa cells. Ku70 siRNA (SiKu70) and Myc-Akt were expressed in HeLa cells as described in (a), and Western blotting analysis was performed with anti-Ku70, anti-Bax, anti-Bcl-2, anti-Myc and anti-actin. d, Akt did not show significant cytoprotective activity in Ku70 null MEFs. Wild type and ku70−/− MEFs (8×10 4 cells/well) were seeded in 12-well plates. Twenty-four h later cells were infected with adenovirus expressing Myc-Myr-Akt. The following day cells were treated with 10 uM Etoposide for additional 12 h and apoptosis was analyzed as described in . e, Expression levels of Ku70, Bax, and Bcl-2 of wild type and ku70−/− MEFs. Wild type and ku70−/− MEFs were analyzed by Western blotting with anti-Ku70 (N3H10), anti-Hdm2 (2A10), anti-p53, anti-Bax (N20), anti-Bcl-2, and anti-actin antibody.

Article Snippet: Immunoprecipitation was performed by incubating 200 uL of the lysates with 2 ug of anti-Bax monoclonal antibody (clone 6A7, BD-Pharmingen) at 4°C for 2 h. Immunocomplexes in 200 uL of the lysates were precipitated with 20 uL protein A-sepharose.

Techniques: Transfection, Infection, Expressing, Staining, Western Blot, Activity Assay

Journal: Cell death and differentiation

Article Title: Hdm2 is an Ubiquitin Ligase of Ku70 -Akt Promotes Cell Survival by Inhibiting Hdm2-dependent Ku70 Destabilization

doi: 10.1038/cdd.2009.6

Figure Lengend Snippet: a, Right panels : Akt maintained Ku70-Bax interaction in etoposide-treated HUVECs. HUVECs were were infected with control adenovirus or adenovirus expressing Myc-Myr-Akt. The next day, cells were treated with 20 uM Etoposide for 8 h. Cells were then lysed and Ku70 (top panel) was co-immunoprecipiatated by anti-Bax polyclonal antibody (detecting both conformationally inactive and active Bax). Activated Bax (second panel) was immunoprecipitated by 6A7 monoclonal antibody (detecting conformationally active Bax). Western blot analysis with anti-Ku70 monoclonal (top and third panels) or anti-Bax polyclonal antibody (N20) (second and fourth pannels) was performed. The levels of Ku70 and Bax in cells in each condition are shown in the third and fourth panels. Left panels: Ku70−/− MEFs were treated as described for Right Panel experiments, and active Bax was immunoprecipitated by 6A7 monoclonal Ab. Immunoprecipitated Bax was detected by Western blot using Bax polyclonal Ab. Ku70 and Bax levels in each sample are shown in the second and third panels, respectively. b, Hypothesized model for Ku70 regulation by Hdm2 and Akt. In the presence of growth/survival factor signals ( Left scheme ), Ku70 levels are high enough to suppress Bax activation. Akt promotes Hdm2 translocation to the nucleus and induces the targeting of p53 for degradation. In the presence of apoptotic signals without active Akt signals ( Right scheme ), Hdm2 remains in the cytosol and it targets Ku70 for degradation. This condition allows the release of Bax from inhibition, and Bax is activated by activators such as BH3 only proteins that are activated by p53.

Article Snippet: Immunoprecipitation was performed by incubating 200 uL of the lysates with 2 ug of anti-Bax monoclonal antibody (clone 6A7, BD-Pharmingen) at 4°C for 2 h. Immunocomplexes in 200 uL of the lysates were precipitated with 20 uL protein A-sepharose.

Techniques: Infection, Expressing, Immunoprecipitation, Western Blot, Activation Assay, Translocation Assay, Inhibition