Journal: Frontiers in Immunology

Article Title: UBE2N as a novel prognostic and therapeutic biomarker of lung adenocarcinoma

doi: 10.3389/fimmu.2025.1636503

Figure Lengend Snippet: Screening of key prognostic genes in LUAD. (A) The Venn diagram for intersecting prognostic genes. (B) The Venn diagram for intersecting common prognostic genes and anti-viral genes. (C–E) Survival curves generated by stratifying patients based on the median expression levels of key genes, UBE2N (C) , USP44 (D) , and SENP7 (E) . (F) Differential expression of key genes in LUAD and paired paracancerous tissues.

Article Snippet: Monoclonal antibodies against UBE2N (A9257) were purchased from Abclonal (Wuhan, China).

Techniques: Generated, Expressing, Quantitative Proteomics

Journal: Frontiers in Immunology

Article Title: UBE2N as a novel prognostic and therapeutic biomarker of lung adenocarcinoma

doi: 10.3389/fimmu.2025.1636503

Figure Lengend Snippet: Validation of the prognostic roles of UBE2N and USP44 across multiple cohorts. (A, B) Survival curves were plotted by grouping patients according to UBE2N expression in the GSE3141 (A) and GSE50081 (B) cohorts. (C, D) Survival curves were plotted by grouping patients according to USP44 expression in the GSE19188 (C) and GSE50081 (D) cohorts. (E, F) DFS and RFS analyses based on UBE2N expression in the GSE30219 (E) and GSE31210 (F) cohorts. (G, H) DFS and RFS analyses based on USP44 expression in the GSE30219 (G) and GSE31210 (H) cohorts.

Article Snippet: Monoclonal antibodies against UBE2N (A9257) were purchased from Abclonal (Wuhan, China).

Techniques: Biomarker Discovery, Expressing

Journal: Frontiers in Immunology

Article Title: UBE2N as a novel prognostic and therapeutic biomarker of lung adenocarcinoma

doi: 10.3389/fimmu.2025.1636503

Figure Lengend Snippet: UBE2N was identified as an independent prognostic indicator of LUAD and correlates with clinicopathologic factors. (A, B) Prognostic independence analyses of key genes and clinicopathologic factors in TCGA-LUAD (A) and GSE30219 (B) cohorts. (C–J) Correlation between the expression of UBE2N and different clinicopathologic factors.

Article Snippet: Monoclonal antibodies against UBE2N (A9257) were purchased from Abclonal (Wuhan, China).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: UBE2N as a novel prognostic and therapeutic biomarker of lung adenocarcinoma

doi: 10.3389/fimmu.2025.1636503

Figure Lengend Snippet: Validation of the association between UBE2N and the clinicopathological progression of LUAD in multiple cohorts. (A, B) Violin plots depict the correlation between UBE2N expression and clinical pathological parameters in cohorts GSE30219 (A) and GSE31210 (B) . (C) Expression levels of UBE2N in histologic grades with different degrees of differentiation in the GSE48465 dataset.

Article Snippet: Monoclonal antibodies against UBE2N (A9257) were purchased from Abclonal (Wuhan, China).

Techniques: Biomarker Discovery, Expressing

Journal: Frontiers in Immunology

Article Title: UBE2N as a novel prognostic and therapeutic biomarker of lung adenocarcinoma

doi: 10.3389/fimmu.2025.1636503

Figure Lengend Snippet: Investigation of the co-expressed gene network and associated functional pathways of UBE2N. (A) Heatmap showing the expression of co-expressed genes of UBE2N. (B) PPI network involving UBE2N and its co-expressed genes. (C) Volcano plot of UBE2N-related differential genes. (D) Functional enrichment analysis of UBE2N-related differential genes. (E, F) KEGG pathways enriched in high-UBE2N (E) and low-UBE2N groups (F) .

Article Snippet: Monoclonal antibodies against UBE2N (A9257) were purchased from Abclonal (Wuhan, China).

Techniques: Functional Assay, Expressing

Journal: Frontiers in Immunology

Article Title: UBE2N as a novel prognostic and therapeutic biomarker of lung adenocarcinoma

doi: 10.3389/fimmu.2025.1636503

Figure Lengend Snippet: UBE2N-related TME and immune functional phenotypes. (A) The violin plot of tumor-infiltrating immune cell abundance. (B) The violin plot of TME scores. (C) The box plot of tumor purity. (D–F) Box plots of the expression of inhibitory immunomodulatory genes (D) , stimulatory immunomodulatory genes (E) , and genes related to antigen presentation and processing (F) .

Article Snippet: Monoclonal antibodies against UBE2N (A9257) were purchased from Abclonal (Wuhan, China).

Techniques: Functional Assay, Expressing, Immunopeptidomics

Journal: Frontiers in Immunology

Article Title: UBE2N as a novel prognostic and therapeutic biomarker of lung adenocarcinoma

doi: 10.3389/fimmu.2025.1636503

Figure Lengend Snippet: Role of UBE2N in tumor immune evasion and immunotherapy. (A–C) Publicly available CRISPR screen data reveal UBE2N as a promoter of tumor immune evasion. (D–F) Role of UBE2N expression in suggesting prognosis in cancer patients receiving immunotherapy. (G) IPS scores in UBE2N subgroups. (H) Role of UBE2N expression in predicting immunotherapy response in different cohorts.

Article Snippet: Monoclonal antibodies against UBE2N (A9257) were purchased from Abclonal (Wuhan, China).

Techniques: CRISPR, Expressing

Journal: Frontiers in Immunology

Article Title: UBE2N as a novel prognostic and therapeutic biomarker of lung adenocarcinoma

doi: 10.3389/fimmu.2025.1636503

Figure Lengend Snippet: Role of UBE2N in cancer chemotherapy and targeted therapy. (A–L) Box plots of differences in drug sensitivity of patients in different UBE2N subgroups to chemotherapeutic and targeted agents including Docetaxel (A) , 5-Fluorouracil (B) , Cisplatin (C) , Cyclophosphamide (D) , MN-64 (E) , Selumetinib (F) , Ibrutinib (G) , Axitinib (H) , SB216763 (I) , Nelarabine (J) , Doramapimod (K) , and Ribociclib (L) . (M–O) Chemical structure diagrams of three potential compounds to inhibit UBE2N.

Article Snippet: Monoclonal antibodies against UBE2N (A9257) were purchased from Abclonal (Wuhan, China).

Techniques:

Journal: Frontiers in Immunology

Article Title: UBE2N as a novel prognostic and therapeutic biomarker of lung adenocarcinoma

doi: 10.3389/fimmu.2025.1636503

Figure Lengend Snippet: Experimental validation of the expression profile and tumor biology of UBE2N. (A) IHC sections reveal high expression of UBE2N in LUAD tissues. (B) IHC sections reveal a correlation between UBE2N expression and tumor diameter. (C) Knockdown of UBE2N expression with siRNA. (D) CCK-8 assay to detect the effect of UBE2N on the proliferative viability of A549 cells. (E–G) Experimental detection of the impact of UBE2N on apoptosis in A549 cells.

Article Snippet: Monoclonal antibodies against UBE2N (A9257) were purchased from Abclonal (Wuhan, China).

Techniques: Biomarker Discovery, Expressing, Knockdown, CCK-8 Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Nonproteolytic ubiquitination regulates chromatin occupancy by the NCoR/SMRT/HDAC3 corepressor complex in MCF-7 breast cancer cells.

doi: 10.1073/pnas.2502805122

Figure Lengend Snippet: Fig. 5. (A) Western blotting with anti-TRAF6 Ab to visualize the nuclear translocation of TRAF6 in MCF-7 cells treated with IL-1β for 5 min. WBs for HDAC2 and β-tubulin were run on the cytoplasmic and nuclear extracts as a control. (B) TRAF6 interaction with NCoR in IL-1β-treated MCF7 cells by immunoprecipitation with anti-NCoR Ab. (C) Graphic representation of the NCoR protein showing the putative TRAF6 interaction motif, and sequence alignment of similar motifs found in human RIP2, IRAK2, and NCoR across species. (D) Interaction between TRAF6 and the identified interaction motif in HEK293T cells transfected with Myc-TRAF6 and wild type (Left) or PxExxDAxAxxA mutant (Right) GAL4-NCoR516-811 plasmids. Immunoprecipitation was performed either with anti-Myc, anti-GAL4, or control IgG, and blotted with anti-GAL4, anti-TRAF6, or anti-Myc Ab. (E) ChIP experiment showing rapid and transient recruitment of TRAF6 and Ubc13 to the BMP7 promoter in response to IL-1β in MCF7 pretreated with E2 for 1 h. (F) Validation of siRNA efficiency in MCF7 cells. Cells are transfected with siRNA (siCtl, siTRAF6, or siUbc13) for 48 h and relative expression measured by RTqPCR, with normalization to b-actin. (G) BMP7 derepression by IL-1β, as measured by RT-PCR, requires both TRAF6 and Ubc13. MCF7 cells transfected with either control, TRAF6, or Ubc13 siRNA, were treated with E2 and/or IL-1β for 6 h. (H) Overexpression of dominant-negative TRAF6 or mutated TAB2 abrogates derepression by IL-1β in U2OS-ERα cells transiently transfected with BMP7promoter reporter and treated with E2 and vehicle (PBS) or IL-1β for 36 h. Luciferase activity was assayed and normalized to b-gal activity. (I) MEKK1’s ubiquitin-interacting motif (UIM) interacts with K63-linked, but not K48-linked, polyubiquitin chains. GST and GST-MEKK1-UIM proteins were expressed in bacteria, purified, and used for GST pulldown with recombinant K63- (Left) or K48-linked (Right) polyubiquitin chains, followed by western blotting with anti-ubiquitin or anti-GST Ab. (J) Ubc13 is required for accumulation of K63-linked polyubiquitin chains and for the recruitment of MEKK1 to the BMP7 promoter in MCF7 cells transfected with control or Ubc13 siRNA and pretreated with E2 for 1 h prior to treatment with IL-1β. ChIP was performed with anti-K63-ubiquitin or anti-MEKK1 Ab or protein A beads alone as control. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01 vs. vehicle control, Student’s t test.

Article Snippet: Commercial antibodies used for western blotting, immunoprecipitation, and ChIP experiments include HDAC3 rabbit polyclonal antibody #ab7030 (Abcam); anti- HDAC3(H- 99) rabbit polyclonal antibody #sc- 11417 (Santa Cruz Biotechnology); anti- ubiquitin mouse monoclonal antibody (P4D1 clone, Cell Signaling Technology); anti- β- tubulin mouse monoclonal antibody #T0198 (Sigma- Aldrich); anti- HDAC2 rabbit polyclonal antibody #ab16032 (Abcam); anti- TAB2 mouse monoclonal antibody sc- 398188, goat polyclonal antibody sc- 11850, and rabbit polyclonal sc- 20756 (Santa Cruz Biotechnology); anti- K63 ubiquitin rabbit monoclonal antibody #05- 1308 (Millipore); anti- TRAF6 rabbit polyclonal antibody sc- 7221 (Santa Cruz Biotechnology); anti- Ubc13 rabbit polyclonal antibody #10243- 1- AP (ProteinTech); anti- Ubc13 (131- 148) rabbit polyclonal antibody #PA1- 41188 (Thermo Scientific); anti- FLAG mouse monoclonal antibody, clone M2, #F3165, and anti- Flag- HRP #A8592 (Sigma- Aldrich); antiMYC(9E10) mouse monoclonal antibody #sc- 40 (Santa Cruz Biotechnology); and anti- GST(56C1) mouse monoclonal antibody #sc- 80998 (Santa Cruz Biotechnology).

Techniques: Western Blot, Translocation Assay, Control, Immunoprecipitation, Sequencing, Transfection, Mutagenesis, Biomarker Discovery, Expressing, Reverse Transcription Polymerase Chain Reaction, Over Expression, Dominant Negative Mutation, Luciferase, Activity Assay, Ubiquitin Proteomics, Bacteria, Purification, Recombinant

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Nonproteolytic ubiquitination regulates chromatin occupancy by the NCoR/SMRT/HDAC3 corepressor complex in MCF-7 breast cancer cells.

doi: 10.1073/pnas.2502805122

Figure Lengend Snippet: Fig. 5. (A) Western blotting with anti-TRAF6 Ab to visualize the nuclear translocation of TRAF6 in MCF-7 cells treated with IL-1β for 5 min. WBs for HDAC2 and β-tubulin were run on the cytoplasmic and nuclear extracts as a control. (B) TRAF6 interaction with NCoR in IL-1β-treated MCF7 cells by immunoprecipitation with anti-NCoR Ab. (C) Graphic representation of the NCoR protein showing the putative TRAF6 interaction motif, and sequence alignment of similar motifs found in human RIP2, IRAK2, and NCoR across species. (D) Interaction between TRAF6 and the identified interaction motif in HEK293T cells transfected with Myc-TRAF6 and wild type (Left) or PxExxDAxAxxA mutant (Right) GAL4-NCoR516-811 plasmids. Immunoprecipitation was performed either with anti-Myc, anti-GAL4, or control IgG, and blotted with anti-GAL4, anti-TRAF6, or anti-Myc Ab. (E) ChIP experiment showing rapid and transient recruitment of TRAF6 and Ubc13 to the BMP7 promoter in response to IL-1β in MCF7 pretreated with E2 for 1 h. (F) Validation of siRNA efficiency in MCF7 cells. Cells are transfected with siRNA (siCtl, siTRAF6, or siUbc13) for 48 h and relative expression measured by RTqPCR, with normalization to b-actin. (G) BMP7 derepression by IL-1β, as measured by RT-PCR, requires both TRAF6 and Ubc13. MCF7 cells transfected with either control, TRAF6, or Ubc13 siRNA, were treated with E2 and/or IL-1β for 6 h. (H) Overexpression of dominant-negative TRAF6 or mutated TAB2 abrogates derepression by IL-1β in U2OS-ERα cells transiently transfected with BMP7promoter reporter and treated with E2 and vehicle (PBS) or IL-1β for 36 h. Luciferase activity was assayed and normalized to b-gal activity. (I) MEKK1’s ubiquitin-interacting motif (UIM) interacts with K63-linked, but not K48-linked, polyubiquitin chains. GST and GST-MEKK1-UIM proteins were expressed in bacteria, purified, and used for GST pulldown with recombinant K63- (Left) or K48-linked (Right) polyubiquitin chains, followed by western blotting with anti-ubiquitin or anti-GST Ab. (J) Ubc13 is required for accumulation of K63-linked polyubiquitin chains and for the recruitment of MEKK1 to the BMP7 promoter in MCF7 cells transfected with control or Ubc13 siRNA and pretreated with E2 for 1 h prior to treatment with IL-1β. ChIP was performed with anti-K63-ubiquitin or anti-MEKK1 Ab or protein A beads alone as control. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01 vs. vehicle control, Student’s t test.

Article Snippet: Commercial antibodies used for western blotting, immunoprecipitation, and ChIP experiments include HDAC3 rabbit polyclonal antibody #ab7030 (Abcam); anti- HDAC3(H- 99) rabbit polyclonal antibody #sc- 11417 (Santa Cruz Biotechnology); anti- ubiquitin mouse monoclonal antibody (P4D1 clone, Cell Signaling Technology); anti- β- tubulin mouse monoclonal antibody #T0198 (Sigma- Aldrich); anti- HDAC2 rabbit polyclonal antibody #ab16032 (Abcam); anti- TAB2 mouse monoclonal antibody sc- 398188, goat polyclonal antibody sc- 11850, and rabbit polyclonal sc- 20756 (Santa Cruz Biotechnology); anti- K63 ubiquitin rabbit monoclonal antibody #05- 1308 (Millipore); anti- TRAF6 rabbit polyclonal antibody sc- 7221 (Santa Cruz Biotechnology); anti- Ubc13 rabbit polyclonal antibody #10243- 1- AP (ProteinTech); anti- Ubc13 (131- 148) rabbit polyclonal antibody #PA1- 41188 (Thermo Scientific); anti- FLAG mouse monoclonal antibody, clone M2, #F3165, and anti- Flag- HRP #A8592 (Sigma- Aldrich); antiMYC(9E10) mouse monoclonal antibody #sc- 40 (Santa Cruz Biotechnology); and anti- GST(56C1) mouse monoclonal antibody #sc- 80998 (Santa Cruz Biotechnology).

Techniques: Western Blot, Translocation Assay, Control, Immunoprecipitation, Sequencing, Transfection, Mutagenesis, Biomarker Discovery, Expressing, Reverse Transcription Polymerase Chain Reaction, Over Expression, Dominant Negative Mutation, Luciferase, Activity Assay, Ubiquitin Proteomics, Bacteria, Purification, Recombinant