antitrim21 af6219  (R&D Systems)

Journal: PLOS Pathogens

Article Title: SARS-CoV-2 Nsp1 suppresses the canonical NF-κB pathway by promoting ubiquitin-dependent degradation of TAK1 kinase

doi: 10.1371/journal.ppat.1014191

Figure Lengend Snippet: a : HEK293T cells were transfected with plasmids expressing FLAG-tagged TAK1 (TAK1-FLAG) and wild-type (WT) or mutant (K48R/K63R) HA-Ub, and 24 h later, were transfected again with a plasmid expressing cMYC-tagged TRIM21 (cMYC-TRIM21) or GFP (GFP-cMYC, Ctrl). Ubiquitinated TAK1 was immunoprecipitated (IP) by anti-FLAG magnetic beads, and the ubiquitination level was analyzed by an anti-HA antibody. GFP (green arrowhead) and TRIM21 (red arrowhead) were detected by anti-cMYC and anti-TAK1 with anti-FLAG antibodies in a-b . The relative signals of HA-Ub and TAK1-FLAG quantified by ImageJ are shown in the right panels in a & e . b : Plasmids expressing cMYC-tagged full-length (FL) or truncated TRIM21 mutants expressing only SPRY or lacking (Δ) the SPRY domain were constructed (left panel). SPRY, SPla and the RYanodine Receptor domain. HEK293T cells were cotransfected with plasmids expressing TAK1-FLAG and FL or truncated cMYC-TRIM21 or GFP. Immunoprecipitation (IP) was performed using anti-cMYC magnetic beads (right panel). c : HEK293T cells were cotransfected with plasmids expressing cMYC-TRIM21 and FLAG-tagged full-length (FL) or truncated N-terminal (1-303 aa, N) or C-terminal (304-606 aa, C) TAK1 mutants. FLAG-tagged GFP (green arrowhead) and TAK1 (blue arrowhead) were detected with an anti-FLAG antibody. d : HEK293T cells were transfected with plasmids expressing HA-tagged TAK1 and cMYC-TRIM21 24 h later and transfected with a plasmid expressing FLAG-tagged Nsp1 (red arrowheads) or a GFP control (Ctrl, green arrowhead). Immunoprecipitation (IP) was performed using anti-HA magnetic beads. The relative signals of cMYC-TRIM21 and TAK1-HA quantified using ImageJ are shown in the right panel. e : HEK293T cells were transfected with a lentiviral vector encoding shRNA targeting TRIM21 (shTRIM21) or luciferase (shLuc) as a control. After puromycin (10 μg/mL) selection for 72 hr, the cells were transfected with an HA-Ub plasmid, and 24 hr later, transfected with an Nsp-cMYC (red arrowheads) or a GFP-cMYC plasmid (Ctrl, green arrowheads). The results in a , d and e are representative of two independent experiments.

Article Snippet: Nsp1 binding kinetics against TAK1 (SinoBiological, #M15-13G) or TRIM21 (MCE, #HY- P71791 ) were measured by surface plasmon resonance (SPR) on a BIAcore 8K+ (Cytiva).

Techniques: Transfection, Expressing, Mutagenesis, Plasmid Preparation, Immunoprecipitation, Magnetic Beads, Ubiquitin Proteomics, Construct, Control, shRNA, Luciferase, Selection

Journal: PLOS Pathogens

Article Title: SARS-CoV-2 Nsp1 suppresses the canonical NF-κB pathway by promoting ubiquitin-dependent degradation of TAK1 kinase

doi: 10.1371/journal.ppat.1014191

Figure Lengend Snippet: In uninfected cells (left), TAB1 binds to TAK1, and the TAK1-TAB1 complex phosphorylates downstream regulators, including IκBα, p38, and Erk, which in turn activate the downstream NF-κB, MAPK and AP-1 pathways. In SARS-CoV-2-infected cells (right), Nsp1 binds to TAK1 at the N-terminal TAB1-binding domain, preventing the formation of the TAK1-TAB1 complex and promoting the binding of TRIM21 to the C-terminus of TAK1. As a result, Nsp1 promotes the proteasomal degradation of TAK1 by enhancing the TRIM21-dependent K48-linked polyubiquitination (Ub) of TAK1, thereby attenuating the activity of the NF-κB and AP-1 pathways. Created in BioRender. Yang, Q. (2026) https://BioRender.com/41xkykd .

Article Snippet: Nsp1 binding kinetics against TAK1 (SinoBiological, #M15-13G) or TRIM21 (MCE, #HY- P71791 ) were measured by surface plasmon resonance (SPR) on a BIAcore 8K+ (Cytiva).

Techniques: Infection, Binding Assay, Activity Assay

Journal: Communications Biology

Article Title: TRIM21 promotes K63-linked ubiquitination of ALKBH5 and suppresses cuproptosis via down-regulation of LIAS in esophageal squamous cell carcinoma

doi: 10.1038/s42003-025-09201-6

Figure Lengend Snippet: A Expression of Tripartite Motif Containing family across TCGA-ESCA datasets. B Expression profiles of TRIM21 across TCGA datasets. C GO enrichment analysis of TRIM21 correlated genes. D Immunoprecipitation of TRIM21 followed by Coomassie blue staining. E Partial mass spectrometry results following TRIM21 co-immunoprecipitation (co-ip). F Co-immunoprecipitation (co-IP) followed by western blot analysis of TRIM21 and ALKBH5. G GST-Pull down of indicated proteins, demonstrating the molecular binding of TRIM21 and ALKBH5 in vitro. H Plasmids overexpressing ALKBH5-Flag, TRIM21-Myc, and HA-UB were transfected into HEK-293T cells and ubiquitination modification was detected by Western Blot. I Transfection of K63- and K48-linked ubiquitination and indicated plasmids followed by Western Blot analysis. J Transfection of plasmids overexpressing truncated RING domain of TRIM21 and indicated plasmids followed by Western Blot analysis. K Molecular docking and predicted binding sites of TRIM21 and ALKBH5 based on protein sequences obtained from the PDB database. L Mass spectrometry analysis suggests that the ubiquitination site of ALKBH5 may be located at K147. M Transfection of plasmids overexpressing ALKBH5 ubiquitination site mutants and indicated plasmids followed by Western Blot analysis. N Nuclear and cytoplasmic fractionation followed by Western blot analysis showing mutation of ALKBH5's K147 ubiquitination site suppressed ALKBH5 translocation to the nucleus (ns: none significant; * p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The primary Abs against human LIAS (11577-1-AP; Proteintech; concentration: 1:1000), human TRIM21 (12108-1-AP; Proteintech; concentration: 1:1000), human ALKBH5 (16837-1-AP; Proteintech; concentration: 1:1000), human IGF2BP3 (A23295; Abclonal; concentration: 1:1000), DYKDDDDK Tag (14793; Cell Signaling Technology; concentration: 1:1000), Myc-Tag (2278; Cell Signaling Technology; concentration: 1:1000) and HA-Tag (3724; Cell Signaling Technology; concentration: 1:1000) were diluted according to the instructions.

Techniques: Expressing, Immunoprecipitation, Staining, Mass Spectrometry, Co-Immunoprecipitation Assay, Western Blot, Binding Assay, In Vitro, Transfection, Ubiquitin Proteomics, Modification, Fractionation, Mutagenesis, Translocation Assay

Journal: Communications Biology

Article Title: TRIM21 promotes K63-linked ubiquitination of ALKBH5 and suppresses cuproptosis via down-regulation of LIAS in esophageal squamous cell carcinoma

doi: 10.1038/s42003-025-09201-6

Figure Lengend Snippet: A Heat map illustrating genes downregulated upon ALKBH5 overexpression detected by RNA sequencing. B KEGG pathway analysis of differentially expressed genes (logFC > 1). C GSEA analysis confirmed significant correlations between RNA degradation pathways and ALKBH5 expression. D RNA Immunoprecipitation (RIP) of ALKBH5 followed by qRT-PCR ( n = 3 independent experiments). E RNA Immunoprecipitation (RIP) of ALKBH5 followed by PCR. F qRT-PCR assays for RNA stability showed that knocking down TRIM21 increased LIAS mRNA stability ( n = 3 independent experiments). G qRT-PCR assays for RNA stability showed that knocking down ALKBH5 increased LIAS mRNA stability ( n = 3 independent experiments). H Measurement of m6A modification levels on LIAS through m6A-IP after knocking down or overexpression of TRIM21 ( n = 3 independent experiments). I Measurement of m6A modification levels on LIAS through m6A-IP after knocking down or overexpression of ALKBH5 ( n = 3 independent experiments). J RIP-qPCR for IGF2BP3 revealed binding of LIAS mRNA to IGF2BP3 ( n = 3 independent experiments). K Knocking down IGF2BP3 notably decreased both the RNA expression levels of LIAS, and this effect was partially rescued by concurrent knockdown of ALKBH5 ( n = 3 independent experiments). L Knocking down IGF2BP3 notably decreased both the protein levels of LIAS, and this effect was partially rescued by concurrent knockdown of ALKBH5 ( n = 3 independent experiments). M qRT-PCR showed LIAS mRNA stability was reduced after IGF2BP3 knockdown ( n = 3 independent experiments). Error bars indicate standard deviations (ns: none significant; * p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The primary Abs against human LIAS (11577-1-AP; Proteintech; concentration: 1:1000), human TRIM21 (12108-1-AP; Proteintech; concentration: 1:1000), human ALKBH5 (16837-1-AP; Proteintech; concentration: 1:1000), human IGF2BP3 (A23295; Abclonal; concentration: 1:1000), DYKDDDDK Tag (14793; Cell Signaling Technology; concentration: 1:1000), Myc-Tag (2278; Cell Signaling Technology; concentration: 1:1000) and HA-Tag (3724; Cell Signaling Technology; concentration: 1:1000) were diluted according to the instructions.

Techniques: Over Expression, RNA Sequencing, Expressing, RNA Immunoprecipitation, Quantitative RT-PCR, Modification, Binding Assay, RNA Expression, Knockdown

Journal: Communications Biology

Article Title: TRIM21 promotes K63-linked ubiquitination of ALKBH5 and suppresses cuproptosis via down-regulation of LIAS in esophageal squamous cell carcinoma

doi: 10.1038/s42003-025-09201-6

Figure Lengend Snippet: A Western blot showing knockdown of TRIM21 significantly increased LIAS expression without affecting ALKBH5 expression. B Nuclear and cytoplasmic fractionation assays followed by Western Blot analysis showed decreased ALKBH5 nuclear localization upon TRIM21 knockdown. C Immunofluorescence staining (IF) demonstrating that knockdown of TRIM21 supresses the nucleocytoplasmic trafficking of ALKBH5. D Apoptosis experiments illustrated that ESCC cells knockdown of TRIM21 or ALKBH5 both increased the sensitivity of cells to cuproptosis, while simultaneous knockout of LIAS attenuated or even counteracted this effect. E CCK8 assays showing that knockdown of TRIM21 inhibited the viability of tumor cells under elesclomol-Cu 2+ stimulation, and this effect could be reversed by LIAS knockdown ( n = 3 independent experiments). F CCK8 assays showing that ESCC cells became more susceptible to elesclomol-Cu 2+ -induced cuproptosis after mutation on ubiquitination site K147 (K147R) compared to Wild Type (WT) ( n = 3 independent experiments). Error bars indicate standard deviations (ns: none significant; * p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The primary Abs against human LIAS (11577-1-AP; Proteintech; concentration: 1:1000), human TRIM21 (12108-1-AP; Proteintech; concentration: 1:1000), human ALKBH5 (16837-1-AP; Proteintech; concentration: 1:1000), human IGF2BP3 (A23295; Abclonal; concentration: 1:1000), DYKDDDDK Tag (14793; Cell Signaling Technology; concentration: 1:1000), Myc-Tag (2278; Cell Signaling Technology; concentration: 1:1000) and HA-Tag (3724; Cell Signaling Technology; concentration: 1:1000) were diluted according to the instructions.

Techniques: Western Blot, Knockdown, Expressing, Fractionation, Immunofluorescence, Staining, Knock-Out, Mutagenesis, Ubiquitin Proteomics

Journal: Communications Biology

Article Title: TRIM21 promotes K63-linked ubiquitination of ALKBH5 and suppresses cuproptosis via down-regulation of LIAS in esophageal squamous cell carcinoma

doi: 10.1038/s42003-025-09201-6

Figure Lengend Snippet: A Subcutaneous xenograft tumor model in nude mice using the esophageal squamous cell carcinoma cell line ECA109, mice were injected with elesclomol in both control (Ctrl) and TRIM21 knockdown (shTRIM21) groups. B Tumor sizes and weights are depicted in the figure ( n = 4 independent experiments). C Proteins from the tumor tissues were extracted for Western Blot analysis, which revealed TRIM21, LIAS protein expression levels. D RNA from the tumor tissues was extracted for qRT-PCR analysis, which revealed TRIM21, LIAS, ALKBH5 mRNA expression levels ( n = 3 independent experiments). E Immunostaining for TRIM21, Ki-67, and TUNEL was performed across the groups. F Quantification of ( E ) ( n = 3 independent experiments). Error bars indicate standard deviations (ns: none significant; * p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The primary Abs against human LIAS (11577-1-AP; Proteintech; concentration: 1:1000), human TRIM21 (12108-1-AP; Proteintech; concentration: 1:1000), human ALKBH5 (16837-1-AP; Proteintech; concentration: 1:1000), human IGF2BP3 (A23295; Abclonal; concentration: 1:1000), DYKDDDDK Tag (14793; Cell Signaling Technology; concentration: 1:1000), Myc-Tag (2278; Cell Signaling Technology; concentration: 1:1000) and HA-Tag (3724; Cell Signaling Technology; concentration: 1:1000) were diluted according to the instructions.

Techniques: Injection, Control, Knockdown, Western Blot, Expressing, Quantitative RT-PCR, Immunostaining, TUNEL Assay

Journal: Communications Biology

Article Title: TRIM21 promotes K63-linked ubiquitination of ALKBH5 and suppresses cuproptosis via down-regulation of LIAS in esophageal squamous cell carcinoma

doi: 10.1038/s42003-025-09201-6

Figure Lengend Snippet: A Co-immunoprecipitation assays revealing that OGT and ALKBH5 can interact with each other. B Myc-OGT plasmid, along with HA-UB and Flag-ALKBH5, was transfected into HEK-293T cells, western blot showed overexpression of myc-OGT promoted the ubiquitination of ALKBH5. C Western blot showing co-transfection of TRIM21 with OGT augmented ALKBH5 ubiquitination. D Co-IP followed by western blot showing overexpression of OGT led to an increase in O-glycosylation of ALKBH5. E Co-IP followed by western blot showing knockdown of OGT led to the decrease in the binding of ALKBH5 and TRIM21, also induced upregulation of LIAS. F Co-IP followed by western blot analysis showing reduced ALKBH5 O-glycosylation and interaction with TRIM21 upon introducing the OGT inhibitor OSMI-1 in different concentrations. G A significant negative correlation in protein expression between the co-expression of OGT and TRIM21 with LIAS, Lipoylated-DLAT (Lip-DLAT), and Lipoylated-DLST (Lip-DLST) expression was observed by western blot, upon OSMI-1 treatment, the expressions of Lip-DLAT, Lip-DLST, and LIAS were restored. H Protein expression showing negative correlation between OGT and TRIM21 with LIAS, Lipoylated-DLAT (Lip-DLAT), and Lipoylated-DLST (Lip-DLST) after OGT or TRIM21 knockdown. I Following treatment with a gradient of OSMI-1 concentrations, nuclear-cytoplasmic fractionation experiments showed that OSMI-1 inhibited ALKBH5's accumulation in the nucleus.

Article Snippet: The primary Abs against human LIAS (11577-1-AP; Proteintech; concentration: 1:1000), human TRIM21 (12108-1-AP; Proteintech; concentration: 1:1000), human ALKBH5 (16837-1-AP; Proteintech; concentration: 1:1000), human IGF2BP3 (A23295; Abclonal; concentration: 1:1000), DYKDDDDK Tag (14793; Cell Signaling Technology; concentration: 1:1000), Myc-Tag (2278; Cell Signaling Technology; concentration: 1:1000) and HA-Tag (3724; Cell Signaling Technology; concentration: 1:1000) were diluted according to the instructions.

Techniques: Immunoprecipitation, Plasmid Preparation, Transfection, Western Blot, Over Expression, Ubiquitin Proteomics, Cotransfection, Co-Immunoprecipitation Assay, Glycoproteomics, Knockdown, Binding Assay, Expressing, Fractionation