hdm2 (2a10)  (Millipore)

Journal: Cell Death Discovery

Article Title: Deciphering UBE4B phosphorylation dynamics: a key mechanism in p53 accumulation and cancer cell response to DNA damage

doi: 10.1038/s41420-025-02441-9

Figure Lengend Snippet: A MCF-7; B A549; C U2OS; D RKO; E RKO-p53 −/− ; F SAOS2. These cells were irradiated with 6 Gy IR and harvested at the indicated time points. UBE4B, HDM2, Wip1, and p53 protein levels were analyzed by immunoblotting. G MCF-7 and RKO cells were treated with 10 J/m 2 UV. These cells were then harvested at the indicated time points. The levels of UBE4B, HDM2, Wip1, and p53 were analyzed by immunoblotting analysis. Actin was used as a loading control. Densitometry was performed using the ImageJ software (NIH), and the relative UBE4B, HDM2, Wip1, and p53 band intensity was normalized to β-actin. An antibody against actin was used as a loading control. Error bars indicate SEM (n-3).

Article Snippet: HDM2 (2A10) , 1:100 , EMD Biosciences , mouse.

Techniques: Irradiation, Western Blot, Control, Software

Journal: Cell Death Discovery

Article Title: Deciphering UBE4B phosphorylation dynamics: a key mechanism in p53 accumulation and cancer cell response to DNA damage

doi: 10.1038/s41420-025-02441-9

Figure Lengend Snippet: A RKO cells were treated with or without 10 J/m2 UV radiation and harvested at 3 hours post-radiation. Cell extracts were immunoprecipitated with p53 (Pab1801) or mouse IgG or p53 antibodies. The levels of UBE4B, p53, and Actin were analyzed by immunoblotting. The dots indicate UV treatment of the samples. For example, lanes 2, 4, and 6 represent UV-treated samples. B RKO cells were transiently transfected with 5, 10, and 20 µg of Flag-tagged Wip1-expressing vector. Thirty-six hours post-transfection, total proteins were extracted and analyzed by immunoblotting with antibodies against Flag (Wip1), UBE4B, and Actin. C H1299 cells were transiently transfected with 5, 10, and 20 µg of Flag-tagged Wip1-expressing vector. Thirty-six hours post-transfection, total proteins were extracted and analyzed by immunoblotting with antibodies against Flag (Wip1), UBE4B, and Actin. D SAOS2 cells were transiently cotransfected with Flag-tagged Wip1 and p53-expressing plasmids. After transfection, cells were treated with or without 6 Gy IR and harvested at 1 and 3 hours. The levels of UBE4B, HDM2, Flag (Wip1), phospho-p53S15, and p53 were detected by immunoblotting. Actin was used as a loading control.

Article Snippet: HDM2 (2A10) , 1:100 , EMD Biosciences , mouse.

Techniques: Immunoprecipitation, Western Blot, Transfection, Expressing, Plasmid Preparation, Control

Journal: Cell Death Discovery

Article Title: Deciphering UBE4B phosphorylation dynamics: a key mechanism in p53 accumulation and cancer cell response to DNA damage

doi: 10.1038/s41420-025-02441-9

Figure Lengend Snippet: A HCT116 cells were irradiated with or without 10 J/m 2 UV and harvested 3 hours post-radiation. Cell extracts (600 to 800 µg of total proteins) were immunoprecipitated with antibodies against UBE4B, Wip1 or IgG [mouse IgG (mIGg) for UBE4B, and rabbit IgG (rIgG) for Wip1]. The levels of UBE4B, Wip1, p53, and phospho-p53S15 were detected by immunoblotting. Approximately 30 to 50 µg of the total proteins used for the immunoprecipitation assay were used as input (direct immunoblotting). B We have created three constructs that express UBE4B and UBE4B mutants. 1- Full-length UBE4B construct. 2- C500 construct that contains only the first 500 amino acids of the N-terminal UBE4B. 3- C800 construct that contains the first 800 amino acids of the N-terminal UBE4B. All constructs are Flag-tagged. C HCT116 cells were transfected with plasmids expressing HDM2, UBE4B, C500 and C800 as indicated. After transfection, cells were irradiated with 10 J/m 2 UV and harvested 3 hours after radiation. Cell extracts were immunoprecipitated with an anti-Flag antibody (M5), and the Wip1 protein was detected by immunoblotting. D HCT116 cells were transfected with plasmids expressing UBE4B, Wip1, C500, and C800 plasmids. Cells were harvested 3 hours after UV treatment and the cell extracts were immunoprecipitated with an anti-p53 antibody (Pab1801). The UBE4B and Wip1 protein bands were detected by immunoblotting with an antibody against Flag. The asterisk (*) denotes the background in Western blots. E SAOS2 cells were transfected with the indicated plasmids. Forty-eight hours later, cells were placed under drug selection (G418) for two weeks. Colonies were fixed with 100% methanol and stained with 0.5% crystal violet. F The ratio of colony formation is presented in the graph. The experiments were carried out in triplicate. ** P < 0.01. G Protein levels of UBE4B (Flag) Wip1 (Flag), and p53 expression were detected by Western blotting with Flag-specific (M5) and p53-specific (Pab1801) antibodies. An antibody against actin was used as a loading control.

Article Snippet: HDM2 (2A10) , 1:100 , EMD Biosciences , mouse.

Techniques: Irradiation, Immunoprecipitation, Western Blot, Construct, Transfection, Expressing, Selection, Staining, Control

Journal: Cell Death Discovery

Article Title: Deciphering UBE4B phosphorylation dynamics: a key mechanism in p53 accumulation and cancer cell response to DNA damage

doi: 10.1038/s41420-025-02441-9

Figure Lengend Snippet: A RKO cells that stably expressed a control siRNA (empty vector) or shWip1 were irradiated with UV and harvested at the indicated time points. Cell lysates were immunoprecipitated with an anti-p53 antibody (Pab1801) or mIgG as a control. The levels of UBE4B, Wip1, HDM2, and p53 were detected by immunoblotting. Knockdown efficiency was further determined by qRT-PCR. Error bars indicate SEM ( n = 3). **** P < 0.0001. Densitometry was performed using the ImageJ software (NIH), and the relative binding affinity between UBE4B-p53 interaction was normalized to IgG and plotted (right panel). The ‘0 h’ timepoint represents samples collected immediately before UV treatment (as non-treated control). B GM03714 and GM0719B cells were irradiated with UV light and harvested at the indicated time points. Cell lysates were immunoprecipitated with an anti-p53 antibody (Pab1801) or mIgG as a control. The levels of UBE4B, phosphorylated UBE4B, p53, and phosphorylated p53 were detected by immunoblotting. Densitometry was performed using the ImageJ software (NIH), and the relative binding affinity between UBE4B-p53 interaction was normalized to IgG and plotted (right panel). The ‘0 h’ timepoint represents samples collected immediately before UV treatment (as non-treated control). C GM03714 and GM0719B cells were irradiated with or without UV light and harvested 3 hours after radiation. The cell lysates were immunoprecipitated with anti-phosphorylated UBE4B antibody (pS669) or rabbit IgG as a control. The expression of ATM and ATR proteins was detected by immunoblotting. D GM0719B cells were preincubated with increased amounts of VE-821 (10–25 µM) or DMSO for 30 min, followed by UV irradiation, and analyzed by immunoblotting. Western blots with indicated antibodies detected the expression of UBE4BS669, UBE4B (total), and actin. E Similarly, GM03714 cells were preincubated with increased KU55933 (10–20 µM) or DMSO for 30 min, followed by UV irradiation, and analyzed by immunoblotting. The expression of UBE4BS669, UBE4B (total), and actin was detected by Western blotting, as indicated. F Wild-type HCT116 and HCT116 CHK2 −/− cells were irradiated with 10 J/m 2 UV. Cells were harvested at 0, 3, 6, and 24 hours after radiation. The levels of phosphorylated UBE4B and phosphorylated p53 were analyzed by immunoblotting. Actin was used as a loading control.

Article Snippet: HDM2 (2A10) , 1:100 , EMD Biosciences , mouse.

Techniques: Stable Transfection, Control, Plasmid Preparation, Irradiation, Immunoprecipitation, Western Blot, Knockdown, Quantitative RT-PCR, Software, Binding Assay, Expressing

Journal: Cell Death Discovery

Article Title: Deciphering UBE4B phosphorylation dynamics: a key mechanism in p53 accumulation and cancer cell response to DNA damage

doi: 10.1038/s41420-025-02441-9

Figure Lengend Snippet: UBE4B mediates HDM2-dependent polyubiquitination of p53 to keep its level low during homeostasis. In response to DNA damage, UBE4B activity is prevented by its phosphorylation mediated by ATR/ATM kinases, which decreases the affinity binding of UBE4B-p53 and leads to the accumulation and activation of p53. However, Wip1 dephosphorylates, stabilizes, and activates UBE4B in response to DNA damage. UBE4B binds to and degrades phosphorylated p53. This figure was created using BioRender.com (granted a license “Academic License Terms” no. ET24RT4ROC).

Article Snippet: HDM2 (2A10) , 1:100 , EMD Biosciences , mouse.

Techniques: Activity Assay, Phospho-proteomics, Binding Assay, Activation Assay

Journal: Cell Death Discovery

Article Title: Deciphering UBE4B phosphorylation dynamics: a key mechanism in p53 accumulation and cancer cell response to DNA damage

doi: 10.1038/s41420-025-02441-9

Figure Lengend Snippet: List of all antibodies.

Article Snippet: HDM2 (2A10) , 1:100 , EMD Biosciences , mouse.

Techniques:

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, HUVECs were seeded in 6-well plates and 24 h later treated with 10 uM Etoposide (Etopo) or 1 uM Doxorubicin (Doxo) for 24 h. After incubation, cells were collected and 2 ug of total protein were examined by Western blotting with anti-Ku70 (N3H10) and anti-actin antibodies. b, HUVECs were plated onto six-well plates at 1.2×10 5 cells per well and transfected with the indicated siRNAs as described in . Results from 24 h and 48 h post-transfection are shown. c, Forty-eight h after siRNA transfection, HUVECs were treated with 10 uM Etoposide for 24 h and 48 h. Apoptosis was detected by nuclear fragmentation using Hoechst-dye nuclear staining. Data shown are averages (± S.E.) (n= 300 cells per condition in triplicate) of three independent experiments. d MEFS ( ku70 +/+ ) were seeded in 6 well dishes and treated with 1 uM Doxorubicin for the times shown in the figure. Cells were then collected and Western blot analysis was performed with anti-Mdm2, anti-p53, anti-Ku70 and anti-Actin antibodies. e, HUVECs were seeded in 6-well plates and 24 h later treated with 1 uM Doxorubicin for the times shown in the figure. After incubation at specific times, cells were collected and examined by Western blotting with anti-Ku70, anti-Hdm2, anti-p53 and anti-actin antibodies.

Article Snippet: Hdm2 antibody (2A10) and (IF2) were purchased from Calbiochem (San Diego, CA).

Techniques: Incubation, Western Blot, Transfection, Staining

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, Hdm2 overexpression decreased Ku70 levels. HUVECs were transfected with pCMV vector, pCMVHdm2-wt or pCMVHdm2-C464S (dead ligase) and cultured for 16 h. Cells were then collected and analyzed by Western blot using anti-Ku70, anti-Hdm2 and anti-Actin antibodies. b, HEK293 cells were transfected with pCMVHdm2-wt and HA-tagged Ubiquitin-wt constructs as indicated. Twenty-one hours later cells were treated with 5 uM MG132 for 3 h; cells were then collected and lysed in RIPA buffer. Immunoprecipitation with anti-Ku70 antibody (H308) was performed as indicated in , and Western blot was done with anti-Ku70 (N3H10) and anti-HA antibody. Mouse IgG was used as control for the immunoprecipitation. c, Ku70 level in mdm2+/+ and mdm2−/− MEFs. Mdm2−/− (p53−/−) and mdm2+/+(p53−/−) MEFs were seeded in 10-cm dishes. Twenty four hours later, cells were harvested and lysed with RIPA buffer. Western blot analysis was then performed with anti-Ku70 (N3H10 antibody). Mdm2 antibody was used to confirm Mdm2 levels. d, In vitro ubiquitination assay of Ku70 by Hdm2. Reaction volumes were prepared as indicated in and incubated for 2 h at 37°C in the presence or absence of 10 uM unlabeled ubiquitin. Reactions were quenched with 50 ul of Laemmli sample buffer containing 2% (v/v) B-mercaptoethanol and boiled for 5 minutes. Western blot was performed with anti-ubiquitin antibody. e, Co-immunoprecipitation of Hdm2 and Ku70. HUVECs were collected and immunoprecipitation with anti-Hdm2 antibody was performed in Chaps buffer. Western blot analysis was done using anti-Ku70 antibody, anti-Ku80 antibody and anti-Hdm2 antibody. f, GST pull-down assay using GST-tagged Hdm2 and recombinant Ku70 was peformed using the ProFound™ pull-down GST protein:protein interaction kit (Pierce) as described by the manufacturer. Interaction was analyzed by Western blotting using anti-Ku70 antibody.

Article Snippet: Hdm2 antibody (2A10) and (IF2) were purchased from Calbiochem (San Diego, CA).

Techniques: Over Expression, Transfection, Plasmid Preparation, Cell Culture, Western Blot, Ubiquitin Proteomics, Construct, Immunoprecipitation, Control, In Vitro, Incubation, Pull Down Assay, Recombinant

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, Nutlin-3 inhibits Ku70-Hdm2 interaction. 50 ng of recombinant Hdm2 were incubated with beads preabsorbed with Hdm2 antibody, for 2 hours. After incubation, 50 ng of recombinant Ku70 (Enzymax) were added in the presence of 20 uM Nutlin-3 when indicated. After additional 2 h incubation, beads were washed and samples were analyzed by Western blotting using anti-Ku70 (H308) or anti-Hdm2 (2A10) antibodies. b, Nutlin-3 inhibited Doxorubicin-induced Ku70 proteolysis. Cells prepared as in (b) were lysed and 2 ug of total protein were analyzed by Western blotting using anti-Ku70 (N3H10) and anti-actin antibodies. c, Nutlin protected HUVECs from Doxorubicin-induced apoptosis. 1×10 5 cells were seeded in 6-well dishes. Twenty-four h later cells were treated with 5 uM Nutlin and after 30 minutes, 1 uM Doxorubicin was added to corresponding wells. Apoptosis was detected twenty-four h later by nuclear fragmentation using Hoechst nuclear staining. Data shown are averages (± S.E.) of triplicated samples (300 cells per condition in triplicate).

Article Snippet: Hdm2 antibody (2A10) and (IF2) were purchased from Calbiochem (San Diego, CA).

Techniques: Recombinant, Incubation, Western Blot, Staining

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, VEGF inhibited doxorubicin-induced apoptosis, HUVECs were serum starved for 15 min and treated with VEGF (100 ng/ml) for 2 hours. Doxorubicin was then added for additional 14 h. Apoptosis was detected by nuclear fragmentation using Hoechst-dye nuclear staining, and b, by caspase activation. Data represent averages (± S.E.) (from 3 independent experiments). c. VEGF inhibited Ku70-proteolysis induced by Doxorubicin. HUVECs were seeded in 6-cm dishes and 24 h later treated with VEGF (100 ng/ml) in serum free media for 2 h. Doxorubicin (1 uM) was then added and cells were collected after additional 16 h incubation. Western blot analysis with anti-Ku70, anti-Hdm2, anti-phospho-Hdm2 (Ser 166, Akt-dependent phosphorylation site), anti-phospho-Akt (Ser 473), anti-total Akt and anti-Actin antibodies was performed . This is representative of three independent experiments.

Article Snippet: Hdm2 antibody (2A10) and (IF2) were purchased from Calbiochem (San Diego, CA).

Techniques: Staining, Activation Assay, Incubation, Western Blot, Phospho-proteomics

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 , Immunohistochemical analysis. HUVECs were seeded on gelatin-coated 35 mm dishes. Cells were serum starved for 15 minutes and treated with VEGF (100 ng/ml) for 2 h. Doxorubicin was then added for 2 h and immunohistochemistry was performed as previously described. After fixation of samples in 4% PFA for 10 minutes and permeabilization with 0.2% Triton X-100, the cellular localization of Hdm2 was detected using a monoclonal antibody against Mdm2 (IF2). After extensive washing in PBS, the samples were further incubated with Texas-Red- conjugated goat anti-mouse IgG and examined under a fluorescent microscope. Arrows identify cells with Hdm2 nuclear localization. b, Quantitative analysis of subcellular localization of Hdm2 detected by immunocytochemistry. In each sample, at least 100 cells were analyzed. Data represent averages (± S.E.) of three independent experiments performed using duplicated samples

Article Snippet: Hdm2 antibody (2A10) and (IF2) were purchased from Calbiochem (San Diego, CA).

Techniques: Immunohistochemical staining, Immunohistochemistry, Incubation, Microscopy, Immunocytochemistry

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: Hdm2 antibody (2A10) and (IF2) were purchased from Calbiochem (San Diego, CA).

Techniques: Transfection, Control, 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: Hdm2 antibody (2A10) and (IF2) were purchased from Calbiochem (San Diego, CA).

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