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: