primary antibodies against trim21  (Proteintech)

Journal: Cell Death Discovery

Article Title: TRIM21 promotes colorectal cancer development through regulating DNA replication by TCF3/MCM2/5 axis

doi: 10.1038/s41420-025-02722-3

Figure Lengend Snippet: A Relative TRIM21 mRNA level in human CRC tissues ( n = 98) and normal intestinal epithelium ( n = 97) from Cohort 1. ** P < 0.01. B Relative TRIM21 mRNA level in human CRC tissues ( n = 20) and normal intestinal epithelium ( n = 20) from the GSE100179 dataset (left panel). Relative TRIM21 mRNA level in COAD (Tumor, n = 275; Normal, n = 349) and READ (Tumor, n = 92; Normal, n = 318) datasets from GAPIA Database (right panel). * P < 0.05. C Representative IHC staining images and immunoreactive scores of TRIM21 from CRC ( n = 87) and adjacent normal tissues ( n = 87) in Cohort 2. Scale bars = 100 μm (upper panel) and 25 μm (lower panel). **** P < 0.0001. D Representative IHC staining images and immunoreactive scores of TRIM21 from CRC tissues at different AJCC stages (Stage Ⅰ, n = 5; Stage Ⅱ, n = 53; Stage Ⅲ, n = 35) in Cohort 2. Scale bars = 100 μm (upper panel) and 25 μm (lower panel). * P < 0.05, ns = no significance. E Kaplan–Meier overall survival curves for all CRC patients ( n = 93) in Cohort 2 based on TRIM21 level. F Kaplan–Meier overall survival curves for CRC patients aged less than 65 years (left panel, n = 37) and those aged 65 years or more (right panel, n = 56) in Cohort 2 based on TRIM21 level.

Article Snippet: Primary antibodies against TRIM21 (12108-1-AP) and TCF3 (67140-1-Ig) were from Proteintech (USA); MCM2 (A23477), MCM5 (A5008) and MKI67 (A20018) were from ABclonal (China); BrdU (ab6326) was from Abcam (UK).

Techniques: Immunohistochemistry

Journal: Cell Death Discovery

Article Title: TRIM21 promotes colorectal cancer development through regulating DNA replication by TCF3/MCM2/5 axis

doi: 10.1038/s41420-025-02722-3

Figure Lengend Snippet: A Cell cycle analysis of CRC cells with or without TRIM21 knockdown by flow cytometry. ** P < 0.01, *** P < 0.001. B Cell proliferation was evaluated by CCK-8 assay in CRC cells with TRIM21 knockdown or overexpression. *** P < 0.001, **** P < 0.0001. C Gene ontology analysis and functional annotation of differentially expressed genes from RNA-seq results between two groups (shNC and shTRIM21, n = 3). These differentially expressed genes were mainly involved in G1-S transition and DNA replication (red boxes). D Detection of mRNA and protein levels of CCND1, CDK4 and CDK6 after TRIM21 knockdown or overexpression in CRC cells by RT-qPCR and western blot, respectively. GAPDH was used as a loading control. ** P < 0.01, *** P < 0.001, **** P < 0.0001. E Detection of MCM2 and MCM5 mRNA levels after TRIM21 knockdown or overexpression in CRC cells by RT-qPCR. ** P < 0.01, *** P < 0.001, **** P < 0.0001. F Detection of MCM2 and MCM5 protein levels after TRIM21 knockdown or overexpression in whole cell lysates, nuclei and chromatin fractions of CRC cells. GAPDH was used as a loading control for whole cell lysates, Lamin B1 and histone H3 were used as loading controls for nuclei and chromatin fractions, respectively.

Article Snippet: Primary antibodies against TRIM21 (12108-1-AP) and TCF3 (67140-1-Ig) were from Proteintech (USA); MCM2 (A23477), MCM5 (A5008) and MKI67 (A20018) were from ABclonal (China); BrdU (ab6326) was from Abcam (UK).

Techniques: Cell Cycle Assay, Knockdown, Flow Cytometry, CCK-8 Assay, Over Expression, Functional Assay, RNA Sequencing, Quantitative RT-PCR, Western Blot, Control

Journal: Cell Death Discovery

Article Title: TRIM21 promotes colorectal cancer development through regulating DNA replication by TCF3/MCM2/5 axis

doi: 10.1038/s41420-025-02722-3

Figure Lengend Snippet: A Fluorescence detection of EdU-positive cells in CRC cells with TRIM21 knockdown or overexpression. Nuclei were stained with DAPI (blue), and replicating DNA was incorporated with EdU (red). Scale bars = 50 μm. ** P < 0.01, *** P < 0.001, **** P < 0.0001. B Detection of EdU-positive cells in CRC cells with TRIM21 knockdown or overexpression by EdU/PI double staining assay. ** P < 0.01. C Cell cycle analysis of HCT8 cells collected at different time points (0 h, 2 h, 4 h, 6 h, 8 h) after release of synchronization from siNC group and siTRIM21 groups by flow cytometry. D Detection of EdU-positive cells in HCT8 cells collected at different time points (0 h, 2 h, 4 h, 6 h, 8 h) after release of synchronization from siNC group and siTRIM21 groups by EdU/PI double staining assay. E Results of DNA fiber assay from HCT8 cells with or without TRIM21 knockdown. The representative images were shown in the left panel. Green fluorescence represented IdU incorporated DNA fibers and red fluorescence represented CIdU incorporated DNA fibers. Scale bars = 5 μm. Analysis of replication fork velocity was presented in the right panel. **** P < 0.0001.

Article Snippet: Primary antibodies against TRIM21 (12108-1-AP) and TCF3 (67140-1-Ig) were from Proteintech (USA); MCM2 (A23477), MCM5 (A5008) and MKI67 (A20018) were from ABclonal (China); BrdU (ab6326) was from Abcam (UK).

Techniques: Fluorescence, Knockdown, Over Expression, Staining, Double Staining, Cell Cycle Assay, Flow Cytometry

Journal: Cell Death Discovery

Article Title: TRIM21 promotes colorectal cancer development through regulating DNA replication by TCF3/MCM2/5 axis

doi: 10.1038/s41420-025-02722-3

Figure Lengend Snippet: A Survival of CRC cells with or without TRIM21 knockdown after treatment with different concentrations of 5-FU or SN-38. ** P < 0.01, *** P < 0.001. B Apoptosis analysis of CRC cells with or without TRIM21 knockdown by 7AAD/Annexin-V staining. * P < 0.05, ** P < 0.01. C Apoptosis analysis of CRC cells with or without TRIM21 knockdown after treatment with a relatively low concentration 5-FU (0.625 μg/mL) or SN-38 (25 nM) by 7AAD/Annexin-V staining. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Primary antibodies against TRIM21 (12108-1-AP) and TCF3 (67140-1-Ig) were from Proteintech (USA); MCM2 (A23477), MCM5 (A5008) and MKI67 (A20018) were from ABclonal (China); BrdU (ab6326) was from Abcam (UK).

Techniques: Knockdown, Staining, Concentration Assay

Journal: Cell Death Discovery

Article Title: TRIM21 promotes colorectal cancer development through regulating DNA replication by TCF3/MCM2/5 axis

doi: 10.1038/s41420-025-02722-3

Figure Lengend Snippet: A Detection of mRNA (left panel) and protein (right panel) levels of TCF3 after TRIM21 knockdown or overexpression in CRC cells, respectively. GAPDH was used as a loading control. ** P < 0.01. B Detection of mRNA (left panel) and protein (right panel) levels of MCM2 and MCM5 after TCF3 overexpression or knockdown in CRC cells, respectively. GAPDH was used as a loading control. ** P < 0.01, *** P < 0.001, **** P < 0.0001. C ChIP assay (upper panel) and ChIP-seq (lower panel) results of TCF3 binding in the promoter regions of MCM2 and MCM5 . ChIP-seq results derived from mouse plasma blast cells and B lymphocytes were obtained from the Cistrome Data Browser. D Cell proliferation was evaluated by CCK-8 assay in CRC cells with TCF3 overexpression or knockdown. **** P < 0.0001. E Fluorescence detection of EdU-positive cells in CRC cells with TCF3 overexpression or knockdown. Nuclei were stained with DAPI (blue), and replicating DNA was incorporated with EdU (red). Scale bars = 50 μm. *** P < 0.001. F Detection of EdU-positive cells in CRC cells with TCF3 overexpression or knockdown by EdU/PI double staining assay. ** P < 0.01, *** P < 0.001. G Survival of HCT8 cells with or without TCF3 overexpression after treatment with different concentrations of 5-FU or SN-38. **** P < 0.0001. H Apoptosis analysis of HCT8 cells with or without TCF3 overexpression after treatment with a relatively low concentration 5-FU (0.625 μg/mL) or SN-38 (25 nM) by 7AAD/Annexin-V staining. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = no significance.

Article Snippet: Primary antibodies against TRIM21 (12108-1-AP) and TCF3 (67140-1-Ig) were from Proteintech (USA); MCM2 (A23477), MCM5 (A5008) and MKI67 (A20018) were from ABclonal (China); BrdU (ab6326) was from Abcam (UK).

Techniques: Knockdown, Over Expression, Control, ChIP-sequencing, Binding Assay, Derivative Assay, Clinical Proteomics, CCK-8 Assay, Fluorescence, Staining, Double Staining, Concentration Assay

Journal: Cell Death Discovery

Article Title: TRIM21 promotes colorectal cancer development through regulating DNA replication by TCF3/MCM2/5 axis

doi: 10.1038/s41420-025-02722-3

Figure Lengend Snippet: A , B Decreased MCM2 and MCM5 mRNA ( A ) and protein ( B ) levels were partially rescued by TCF3 knockdown after TRIM21 downregulation in HCT8 cells. GAPDH was used as a loading control. ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = no significance. C After TRIM21 knockdown, TCF3 downregulation partially reversed the proliferation restriction in HCT8 cells. ** P < 0.01, *** P < 0.001. D , E TCF3 knockdown restored the decrease in the percentage of EdU-positive HCT8 cells caused by TRIM21 downregulation to a certain extent. Figure 6D showed the results of fluorescence detection. Nuclei were stained with DAPI (blue), and replicating DNA was incorporated with EdU (red). Scale bars = 50 μm. Figure 6E displayed the results of detection by EdU/PI double staining assay. ** P < 0.01, *** P < 0.001, **** P < 0.0001. F The increased apoptosis of HCT8 cells caused by TRIM21 knockdown after 5-FU (0.625 μg/mL) or SN-38 (25 nM) treatment was rescued by TCF3 downregulation to a certain extent. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Primary antibodies against TRIM21 (12108-1-AP) and TCF3 (67140-1-Ig) were from Proteintech (USA); MCM2 (A23477), MCM5 (A5008) and MKI67 (A20018) were from ABclonal (China); BrdU (ab6326) was from Abcam (UK).

Techniques: Knockdown, Control, Fluorescence, Staining, Double Staining

Journal: Cell Death Discovery

Article Title: TRIM21 promotes colorectal cancer development through regulating DNA replication by TCF3/MCM2/5 axis

doi: 10.1038/s41420-025-02722-3

Figure Lengend Snippet: A Representative images of tumors from nude mice ( n = 6) inoculation with lentivirus stably transfected HCT116 cells with or without 5-FU intervention. B Analysis performed on the volume and weight of the tumors from Fig. 7A. ** P < 0.01, *** P < 0.001, **** P < 0.0001. C Representative IHC staining images and corresponding immunoreactive scores of TRIM21, MCM2, MCM5, TCF3, BrdU and MKI67 of the tumors from Fig. 7A. Scale bars = 50 μm (upper panel) and 20 μm (lower panel). ** P < 0.01, *** P < 0.001, **** P < 0.0001. D Schematic diagram of TRIM21 regulating DNA replication in CRC cells.

Article Snippet: Primary antibodies against TRIM21 (12108-1-AP) and TCF3 (67140-1-Ig) were from Proteintech (USA); MCM2 (A23477), MCM5 (A5008) and MKI67 (A20018) were from ABclonal (China); BrdU (ab6326) was from Abcam (UK).

Techniques: Stable Transfection, Transfection, Immunohistochemistry

Journal: Cell Biology and Toxicology

Article Title: Neutrophil-derived heparin-binding protein increases endothelial permeability in acute lung injury by promoting TRIM21 and the ubiquitination of P65

doi: 10.1007/s10565-025-10005-x

Figure Lengend Snippet: HBP interacts with TRIM21 and increases its protein stability. A Co-IP detection of HBP-TRIM21 binding in ECs treated with purified HBP. B Immunofluorescence showing co-localization of TRIM21 and HBP in the cytoplasm of ECs, scale bar: 10 µm. C,D mRNA and protein levels of TRIM21 in ECs overexpressing HBP. Data are represented as mean ± SD, n = 3, ** P < 0.01, ns P > 0.05. E CHX treatments to analyze TRIM21 stability and degradation in HBP-overexpressing ECs. Data are represented as mean ± SD, n = 3, ** P < 0.01. F Effects of MG132 on TRIM21 expression in HBP overexpression ECs. Data are represented as mean ± SD, n = 3, ** P < 0.01. G Ubiquitination levels of TRIM21 in HBP overexpression ECs treated with MG132 (10 µM) for 12 h. Data are represented as mean ± SD, n = 3, *** P < 0.001. H HEK293T cells were co-transfected with either vector or Flag-HBP together with HA-Ub WT, and its mutants (HA-Ub K48 and HA-Ub K63) and treated with MG132 (20 µM) for 12 h to assess TRIM21 ubiquitination levels. Data are represented as mean ± SD, n = 3,** P < 0.01,*** P < 0.001

Article Snippet: Cells were incubated with primary antibodies against HBP (MAB461Hu24,Cloud-clone,1:100) and TRIM21(12,108–1-AP, proteintech,1:100) at 4 °C overnight.

Techniques: Co-Immunoprecipitation Assay, Binding Assay, Purification, Immunofluorescence, Expressing, Over Expression, Ubiquitin Proteomics, Transfection, Plasmid Preparation

Journal: Cell Biology and Toxicology

Article Title: Neutrophil-derived heparin-binding protein increases endothelial permeability in acute lung injury by promoting TRIM21 and the ubiquitination of P65

doi: 10.1007/s10565-025-10005-x

Figure Lengend Snippet: HBP promotes the interaction between TRIM21 and P65, and regulates endothelial cell permeability and glycolysis through TRIM21-induced ubiquitination of P65. A The effect of recombinant HBP and sepsis-derived PMNs on the interaction between TRIM21 and P65. Data are represented as mean ± SD, n = 3, * P < 0.05, ** P < 0.01,*** P < 0.001. B, C Impact of HBP overexpression and TRIM21 knockdown on P65 phosphorylation and nuclear translocation. Data are represented as mean ± SD, n = 3, * P < 0.05, ** P < 0.01, *** P < 0.001. D, E The effects of HBP overexpression and knockdown TRIM21 on TEER values and FITC-dextran measurements. F, G Western blot detects the effects of HBP overexpression and knockdown TRIM21 on tight junction protein expression in ECs. Data are represented as mean ± SD, n = 3, * P < 0.05, *** P < 0.001. H-J ECAR, lactate production, and glycolysis in ECs with altered HBP and TRIM21 expression. Data are represented as mean ± SD, n = 3, ** P < 0.01, *** P < 0.001

Article Snippet: Cells were incubated with primary antibodies against HBP (MAB461Hu24,Cloud-clone,1:100) and TRIM21(12,108–1-AP, proteintech,1:100) at 4 °C overnight.

Techniques: Permeability, Ubiquitin Proteomics, Recombinant, Derivative Assay, Over Expression, Knockdown, Phospho-proteomics, Translocation Assay, Western Blot, Expressing

Journal: Cell Biology and Toxicology

Article Title: Neutrophil-derived heparin-binding protein increases endothelial permeability in acute lung injury by promoting TRIM21 and the ubiquitination of P65

doi: 10.1007/s10565-025-10005-x

Figure Lengend Snippet: In vivo experiments confirm that silencing TRIM21 reverses HBP-induced lung injury. A, TRIM21 and P65 expression in the lung primary endothelial cells from mice treated with HBP. Data are represented as mean ± SD, n = 5, *** P < 0.001,ns, not significant. B,C Ubiquitination levels of TRIM21 and P65 in the lung tissues of mice treated with HBP. D Neutrophil count in BALF of mice treated with HBP. Data are represented as mean ± SD, n = 5, *** P < 0.001. E TRIM21 expression in the lung primary endothelial cells of sh-NC and sh-TRIM21 mice detected by Western blot. Data are represented as mean ± SD, n = 5, *** P < 0.001. F,G Lung tissues HE staining and lung inflammation score of sh-NC and sh-TRIM21 mice following HBP exposure, scale bar: 50 µm. Data are represented as mean ± SD, n = 5, *** P < 0.001. H-J Measurements of lung W/D ratio, Evans Blue leakage, and BALF lactate in sh-NC and sh-TRIM21 mice exposed to HBP. Data are represented as mean ± SD, n = 5, ** P < 0.01, *** P < 0.001

Article Snippet: Cells were incubated with primary antibodies against HBP (MAB461Hu24,Cloud-clone,1:100) and TRIM21(12,108–1-AP, proteintech,1:100) at 4 °C overnight.

Techniques: In Vivo, Expressing, Ubiquitin Proteomics, Western Blot, Staining