Journal: Advanced Science

Article Title: Macrophage TRIM21 Inhibition Ameliorates Murine Acute Pancreatitis via PHB2‐Mediated Mitochondrial Stabilization

doi: 10.1002/advs.202517877

Figure Lengend Snippet: TRIM21 expression was elevated in acute pancreatitis (AP). (A) Cross‐dataset analysis of TRIM family gene expression using three independent Gene Expression Omnibus (GEO) datasets. (B) Immunohistochemical (IHC) staining for TRIM21 in the pancreas from L‐arginine‐treated and saline‐treated mice (scale bar: 100 µm; n = 6). (C, D) Co‐immunofluorescence of TRIM21 (red) with F4/80 (green; macrophage marker) or MPO (green; neutrophil marker). Cell nuclei were stained with DAPI (scale bar: 25 µm; n = 5). (E) Representative Western blotting images (upper panel) and quantification (lower panel) of the expression levels of TRIM21 protein in circulating monocytes from L‐arginine‐treated and saline‐treated mice ( n = 6). (F) The plasma levels of TRIM21 in healthy controls ( n = 29) and those with mild AP ( n = 41), moderately severe AP ( n = 37), and severe AP ( n = 23). (G) Representative Western blotting images (upper panel) and quantification (lower panel) of TRIM21 levels in circulating monocytes from patients with AP ( n = 12 per subtype) and healthy controls ( n = 12). (H) RT‐qPCR analysis of the mRNA levels of TRIM21 ( n = 5) and (I) Western blotting of the protein levels of TRIM21 ( n = 6) in bone marrow‐derived macrophages (BMDMs) treated with acinar cell supernatants (ACS). Data are expressed as mean ± SEM (B, E–I), and statistical analyses were conducted using Welch's t‐test (B, H), Student's t‐test (E, I), Kruskal‐Wallis test with Dunn's post hoc test (F), and ANOVA with Tukey HSD post hoc test (G).

Article Snippet: TRIM21 was activated using vilazodone (25 mg/kg; MedChemExpress) and downregulated using quisinostat (10 mg/kg; MedChemExpress).

Techniques: Expressing, Gene Expression, Immunohistochemical staining, Immunohistochemistry, Saline, Immunofluorescence, Marker, Staining, Western Blot, Clinical Proteomics, Quantitative RT-PCR, Derivative Assay

Journal: Advanced Science

Article Title: Macrophage TRIM21 Inhibition Ameliorates Murine Acute Pancreatitis via PHB2‐Mediated Mitochondrial Stabilization

doi: 10.1002/advs.202517877

Figure Lengend Snippet: Macrophage Trim21 deficiency attenuated L‐arginine‐induced AP. (A–G) Trim21 M‐KO and Trim21 M‐WT mice were treated with either saline or L‐arginine for 72 h ( Trim21 M‐WT + Saline, Trim21 M‐WT + L‐arginine, Trim21 M‐KO + Saline, Trim21 M‐KO + L‐arginine; n = 10 per group). (A) Serum amylase levels. (B) Representative gross morphology of the pancreas. (C) Pancreas‐to‐body weight ratio. (D) Hematoxylin and eosin (H&E) staining (upper panel) and histopathological scoring (lower panel) of the pancreatic tissue. (E) Representative images (upper panel) and quantification (lower panel) of IHC staining for interleukin‐1 beta (IL‐1β) / tumor necrosis factor‐alpha (TNF‐α) in the pancreatic tissue. (F, G) IL‐1β and TNF‐α levels in serum samples and bronchoalveolar lavage fluid (BALF). Data are expressed as mean ± SEM (A, C–G), and statistical analyses were conducted using ANOVA with Tukey HSD post hoc test (A, TNF‐α in E), Welch's ANOVA with Games‐Howell post hoc test (C, IL‐1β in E, F, G), and Kruskal‐Wallis test with Dunn's post hoc test (D).

Article Snippet: TRIM21 was activated using vilazodone (25 mg/kg; MedChemExpress) and downregulated using quisinostat (10 mg/kg; MedChemExpress).

Techniques: Saline, Staining, Immunohistochemistry

Journal: Advanced Science

Article Title: Macrophage TRIM21 Inhibition Ameliorates Murine Acute Pancreatitis via PHB2‐Mediated Mitochondrial Stabilization

doi: 10.1002/advs.202517877

Figure Lengend Snippet: Vilazodone‐mediated activation of TRIM21 exacerbated L‐arginine‐induced AP. (A‐G) Trim21 M‐WT mice pretreated with vehicle or vilazodone intraperitoneally received saline or L‐arginine for 72 h (Vehicle + Saline, Vehicle + L‐arginine, Vilazodone + Saline, Vilazodone + L‐arginine; n = 10 per group). (A) Serum amylase levels. (B) Representative gross morphology of the pancreas. (C) Pancreas‐to‐body weight ratio. (D) H&E staining (upper panel) and histopathological scores (lower panel) of the pancreatic tissue. (E) Representative images (upper panel) and quantification (lower panel) of IHC staining for IL‐1β and TNF‐α in pancreatic tissue. (F, G) IL‐1β and TNF‐α levels in serum samples and BALF. Data are expressed as mean ± SEM (A, C–G), and statistical analyses were conducted using ANOVA with Tukey HSD post hoc test (A, C), Kruskal‐Wallis test with Dunn's post hoc test (D), and Welch's ANOVA with Games‐Howell post hoc test (E–G).

Article Snippet: TRIM21 was activated using vilazodone (25 mg/kg; MedChemExpress) and downregulated using quisinostat (10 mg/kg; MedChemExpress).

Techniques: Activation Assay, Saline, Staining, Immunohistochemistry

Journal: Advanced Science

Article Title: Macrophage TRIM21 Inhibition Ameliorates Murine Acute Pancreatitis via PHB2‐Mediated Mitochondrial Stabilization

doi: 10.1002/advs.202517877

Figure Lengend Snippet: Macrophage TRIM21 regulates PHB2‐mediated cGAS/STING signaling pathways. (A) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed proteins (DEPs) in pancreatic tissues from L‐arginine‐induced Trim21 M‐WT and Trim21 M‐KO mice ( n = 3). The bubble diagram represents the number of genes under a specific term. The color of the dots represents the p ‐value. (B, C) Representative Western blotting images and quantification of the expression levels of mitophagy‐associated proteins (PINK1, Parkin, p62, and LC3B) in BMDMs from Trim21 M‐WT and Trim21 M‐KO mice, or Trim21 M‐WT mice transfected with an adenovirus carrying Trim21 (Ad‐ Trim21 ) and then stimulated with ACS for 12 h ( n = 5). (D) Co‐immunoprecipitation (co‐IP) with liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) analysis of pancreatic tissues from L‐arginine‐induced AP and saline‐induced control, with anti‐TRIM21 antibody or IgG antibody ( n = 3). (E) Intersection analysis of the DEPs associated with mitophagy and cytosolic DNA‐sensing pathways with the exclusive LC‐MS/MS signature proteins. (F, G) Western blotting of PHB2 in BMDMs from Trim21 M‐WT and Trim21 M‐KO mice, or Trim21 M‐WT mice transfected with Ad‐ Trim21 and then stimulated with ACS for 12 h ( n = 5). (H) Representative immunofluorescence images of mitophagic events in BMDMs from Trim21 M‐WT or Trim21 M‐KO mice after 12 h of treatment with ACS. Mitochondria (TOMM20, red) colocalizing with the autophagic marker LC3B (visualized using adenoviral mCherry‐GFP‐LC3B fusion protein) were identified as mitophagic events (indicated by the red arrows; scale bar: 10 µm; n = 5). (I) Representative immunofluorescence images of cytosolic mtDNA accumulation in BMDMs from Trim21 M‐WT or Trim21 M‐KO mice after 12 h of treatment with ACS. Areas where TFAM (mitochondrial transcription factor A, green) and double‐stranded DNA (dsDNA, red) signals overlapped, but did not co‐localize with mitochondria (TOMM20, yellow) or the nucleus (DAPI, blue), were identified as cytosolic mtDNA (indicated by the red arrows; scale bar: 10 µm; n = 5). (J) RT‐qPCR of cytosolic mtDNA in BMDMs from Trim21 M‐WT or Trim21 M‐KO mice after 12 h of treatment with ACS ( n = 5). (K) Representative Western blot images (left panel) and quantification (right panel) of mitophagy‐associated protein levels (PINK1, Parkin, p62, and LC3B) in BMDMs from Trim21 M‐WT or Trim21 M‐KO mice following transfection with PHB2 siRNA (si‐ Phb2 ) and subsequent 12‐h ACS treatment ( n = 5). (L) Representative immunofluorescence images of mitophagic events in BMDMs from Trim21 M‐WT or Trim21 M‐KO mice after transfection with Phb2 siRNA (si‐ Phb2 ) and 12 h of treatment with ACS. TOMM20 (red) colocalizing with the autophagic marker LC3B (visualized using adenoviral mCherry‐GFP‐LC3B fusion protein) was identified as a mitophagic event (indicated by the red arrows; scale bar: 10 µm; n = 5). (M) Representative immunofluorescence images (indicated by the red arrows; scale bar: 10 µm; n = 5) and (N) RT‐qPCR ( n = 5) of cytosolic mtDNA in BMDMs from Trim21 M‐WT or Trim21 M‐KO mice after transfection with si‐ Phb2 and 12 h of treatment with ACS. Data are expressed as mean ± SEM (B, C, F, G, J, K, N). Statistical analyses were conducted using ANOVA with Tukey HSD post hoc test (indicators other than p62 in B, C, F, G, K, N), Welch's ANOVA with Games‐Howell post hoc test (p62 in B), and Student's t‐test (J).

Article Snippet: TRIM21 was activated using vilazodone (25 mg/kg; MedChemExpress) and downregulated using quisinostat (10 mg/kg; MedChemExpress).

Techniques: Protein-Protein interactions, Western Blot, Expressing, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Saline, Control, Immunofluorescence, Marker, Quantitative RT-PCR

Journal: Advanced Science

Article Title: Macrophage TRIM21 Inhibition Ameliorates Murine Acute Pancreatitis via PHB2‐Mediated Mitochondrial Stabilization

doi: 10.1002/advs.202517877

Figure Lengend Snippet: TRIM21 could bind to PHB2 via its coiled‐coil domain and promoted K11‐linked ubiquitination‐dependent proteasomal degradation. (A) Co‐IP with anti‐TRIM21 (left) or anti‐PHB2 (right) in BMDMs (n = 5). (B) Co‐localization of TRIM21 (red) with PHB2 (green) in BMDMs. Cell nuclei were stained with DAPI (blue) (scale bar: 10 µm; n = 5). (C) Domain diagrams of TRIM21 WT and deletion mutants. (D) Co‐IP analysis of the interactions of Flag‐PHB2 with His‐TRIM21 (WT or mutants) in HEK293T cells ( n = 5). (E) Representative Western blotting images (left panel) and quantification (right panel) of PHB2 levels in BMDMs transfected with gradient concentrations of Ad‐ Trim21 ( n = 5). (F) Representative Western blotting images (upper panel) and quantification (lower panel) of PHB2 levels in Ad‐ Trim21 ‐expressing BMDMs pretreated with chloroquine (CQ; 50 µ m ), 3‐methyladenine (3‐MA; 10 m m ), and Z‐Leu‐Leu‐Leu‐al (MG132; 10 µ m ) for 12 h ( n = 5). (G) Representative Western blotting images (upper panel) and quantification (lower panel) of PHB2 levels in BMDMs from Trim21 M‐WT or Trim21 M‐KO mice treated with cycloheximide (CHX; 100 µg/mL) or MG132 (10 µ m ) for 0 h or 12 h ( n = 5). (H) Co‐IP results for PHB2 ubiquitination levels. BMDMs from Trim21 M‐WT or Trim21 M‐KO mice were treated with MG132 (10 µ m ) for 12 h and with ACS for 12 h ( n = 5). (I) Co‐IP results for exogenous PHB2 ubiquitination levels. HEK293T cells were transfected with the control vector, His‐TRIM21(WT), Flag‐PHB2, and MYC‐ubiquitin (WT/K11/K48/K63) ( n = 5). Data are expressed as mean ± SEM (E–G). Statistical analyses were conducted using ANOVA with Tukey HSD post hoc test (E, F) and Kruskal‐Wallis test with Dunn's post hoc test (G).

Article Snippet: TRIM21 was activated using vilazodone (25 mg/kg; MedChemExpress) and downregulated using quisinostat (10 mg/kg; MedChemExpress).

Techniques: Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Staining, Western Blot, Transfection, Expressing, Control, Plasmid Preparation

Journal: Advanced Science

Article Title: Macrophage TRIM21 Inhibition Ameliorates Murine Acute Pancreatitis via PHB2‐Mediated Mitochondrial Stabilization

doi: 10.1002/advs.202517877

Figure Lengend Snippet: Macrophage Phb2 deficiency reversed Trim21 deficiency‐mediated improvement in AP. (A–H) Trim21 M‐KO and Trim21 M‐WT mice received AAV9‐sh Phb2 or AAV9‐sh NC for 4 weeks, followed by 72 h of infusion of L‐arginine ( Trim21 M‐WT + AAV9‐sh NC + L‐arginine, Trim21 M‐WT + AAV9‐sh Phb2 + L‐arginine, Trim21 M‐KO + AAV9‐sh NC + L‐arginine, Trim21 M‐KO + AAV9‐sh Phb2 + L‐arginine, n = 10 per group). (A) Serum amylase levels. (B) Gross morphology of the pancreas. (C) Pancreas‐to‐body weight ratio. (D) H&E staining (upper panel) and histopathological scores (lower panel) of the pancreas. Representative images (E) and quantification (F) of IHC staining for IL‐1β and TNF‐α in the pancreatic tissue. (G, H) Serum and BALF levels of IL‐1β and TNF‐α. Data are expressed as mean ± SEM (A, C, D, F–H), and statistical analyses were conducted using Welch's ANOVA with Games‐Howell post hoc test (A, F), ANOVA with Tukey HSD post hoc test (C, G, H), and Kruskal‐Wallis test with Dunn's post hoc test (D).

Article Snippet: TRIM21 was activated using vilazodone (25 mg/kg; MedChemExpress) and downregulated using quisinostat (10 mg/kg; MedChemExpress).

Techniques: Staining, Immunohistochemistry

Journal: Advanced Science

Article Title: Macrophage TRIM21 Inhibition Ameliorates Murine Acute Pancreatitis via PHB2‐Mediated Mitochondrial Stabilization

doi: 10.1002/advs.202517877

Figure Lengend Snippet: TRIM21 suppression mitigated L‐arginine‐induced development of AP. (A–I) Trim21 M‐WT mice pretreated with vehicle or quisinostat intraperitoneally received L‐arginine for 72 h (AP, quisinostat + AP; n = 10 per group). (A) Serum amylase levels. (B) Gross morphology of the pancreas. (C) Pancreas‐to‐body weight ratio. H&E staining (D) and histopathological scores (E) of the pancreas. Representative images (F) and quantification (G) of IHC staining for IL‐1β and TNF‐α in pancreatic tissue. (H, I) IL‐1β and TNF‐α levels in serum samples and BALF. Data are expressed as mean ± SEM (A, C, E, G–I), and statistical analyses were conducted using Student's t‐test (A, C, IL‐1β in G, TNF‐α in I), Mann‐Whitney U test (E, H, IL‐1β in I), and Welch's t‐test (TNF‐α in G).

Article Snippet: TRIM21 was activated using vilazodone (25 mg/kg; MedChemExpress) and downregulated using quisinostat (10 mg/kg; MedChemExpress).

Techniques: Staining, Immunohistochemistry, MANN-WHITNEY

Journal: Advanced Science

Article Title: Macrophage TRIM21 Inhibition Ameliorates Murine Acute Pancreatitis via PHB2‐Mediated Mitochondrial Stabilization

doi: 10.1002/advs.202517877

Figure Lengend Snippet: Proposed working model of TRIM21. Macrophage‐specific TRIM21 upregulation exacerbates the development of AP. Mechanistically, macrophage TRIM21 facilitates PHB2 degradation through ubiquitin‐proteasome processing, leading to cytosolic mtDNA accumulation via impaired PHB2‐mediated mitophagy. This mtDNA homeostasis disruption triggers cGAS‐STING axis activation, thereby exacerbating inflammatory cascades during AP progression.

Article Snippet: TRIM21 was activated using vilazodone (25 mg/kg; MedChemExpress) and downregulated using quisinostat (10 mg/kg; MedChemExpress).

Techniques: Ubiquitin Proteomics, Disruption, Activation Assay

Journal: Oncogene

Article Title: TAF1 acetyltransferase promotes colorectal carcinoma metastasis by catalyzing β-hydroxybutyrylation of KCTD9

doi: 10.1038/s41388-025-03644-1

Figure Lengend Snippet: A Tubacin decreases KCTD9 protein and increases KCTD9 K123bhb and K129bhb levels in HEK-293T and different CRC cell lines. HEK293T, HCT116, and LOVO cells were treated with Tubacin for the indicated times. Cell lysates were immunoprecipitated with anti-KCTD9 and analyzed by WB. B Tubacin has a negligible effect on KCTD9 mRNA levels. HEK293T, HCT116, and LOVO cells were treated with Tubacin for the indicated times. The relative KCTD9 mRNA levels were determined by qPCR. C , D Immunoblot and qPCR analysis of the KCTD9 K123bhb and K129bhb ( C ), and KCTD9 mRNA levels ( D ) in control and TAF1-knockdown HCT116 and LOVO cells. E Co-IP of endogenous TRIM21 with anti KCTD9 antibody in HCT116 cells. F Effects of TAF1 WT or HAT activity-deficient mutant Δ844-850 on KCTD9 association with TRIM21. Myc-TRIM21 and indicated Flag-TAF1 were co-transfected into HEK-293T cells with HA-KCTD9 constructs or empty vector control (EV). G Flag-tagged TRIM21 protein ubiquitinated HA-tagged KCTD9 analyzed by in vitro ubiquitination assays using purified recombinant proteins. Purified recombinant TRIM21 and TRIM21 ΔRING protein were incubated with ATP, E1, E2 proteins, ubiquitin, KCTD9-WT peptide and KCTD9-Kbhb peptide (10 μM or 20 μM) along with purified recombinant HA-tagged KCTD9 protein. H , I Effects of ectopic expression of KCTD9 WT or KCTD9 2KR mutant on KCTD9 protein degradation in HCT116 cells co-transfection of Myc-TRIM21 and Flag-TAF1 in exposure to cycloheximide (CHX, 100 μM) treatment (0, 30, 60, and 90 min). J Effects of KCTD9 WT, 2KQ or 2KR mutant on KCTD9 ubiquitination in HCT116/sgKCTD9 cells co-transfection of Myc-TRIM21 or/and Flag-TAF1. Data are representative of two or three independent experiments with similar results. Error bars, ± SD. * P < 0.05 or ** P < 0.01, by paired two-way Student’s t-test. n.s., negative significant.

Article Snippet: Antibodies for TAF1 (#12781), HES1 (#11988), CXCR4 (#64837), CXCL12 (#3740), activated Notch1 (NICD, #4380), CBP (#7389 T), P300 (#86377), TRIM21 (#92043), HA (#3724), Actin (#4967S) and His (#12698) were from CST (Danvers, MA); anti-β-Hydroxybutyryllysine (#PTM-1201RM) was from PTM BIO (Chicago, USA); antibody for KCTD9 (ab180937) were from Abcam Biotechnology (Abcam, Cambridge, UK); HEY1 (#19929-1-AP), MYC (#10828-1-AP), SNAI1 (#13099-1-AP) were from Proteintech; and anti-KCTD9 K123bhb and K129bhb rabbit polyclonal antibody generated by Proteintech using the peptide DKGVWGN-K(bhb)-QDHRGAF and D-K(bhb)-GVWGNK; and KCTD9-Kbhb peptide was synthesized by using the peptide of DK(bhb)GVWGN-K(bhb)-QDHRGAF; an antibody for FLAG (F3165) was from Sigma-Aldrich; antibodies for GAPDH (sc-25778) and GST (sc-138) were from Santa Cruz Biotechnology (Santa Cruz, CA); the secondary antibodies, anti-rabbit IgG, HRP-linked antibody (#7074) and anti-mouse IgG, HRP-linked antibody (#7076) were purchased from CST (Danvers, MA).

Techniques: Immunoprecipitation, Western Blot, Control, Knockdown, Co-Immunoprecipitation Assay, Activity Assay, Mutagenesis, Transfection, Construct, Plasmid Preparation, In Vitro, Ubiquitin Proteomics, Purification, Recombinant, Incubation, Expressing, Cotransfection

Journal: Oncogene

Article Title: TAF1 acetyltransferase promotes colorectal carcinoma metastasis by catalyzing β-hydroxybutyrylation of KCTD9

doi: 10.1038/s41388-025-03644-1

Figure Lengend Snippet: A Representative images of TAF1, KCTD9 K123bhb, KCTD9 K129bhb and NICD expressions in 113 clinical CRC primary tumor tissues. Scale bars, 50 μm. B Correlation of expression levels between TAF1, KCTD9 K123bhb, KCTD9 K129bhb and NICD in ( A ). C Prognosis comparison of CRC patients with TAF1/KCTD9-K123bhb, TAF1/KCTD9-K129bhb and TAF1/NICD ectopic differential expression using Kaplan-Meier survival analysis. D A working model of TAF1 promoted CRC metastasis through the KCTD9 Kbhb modification status. TAF1 directly binds to and catalyzes KCTD9 Kbhb modification at lys123 and lys129, thereby enhancing KCTD9 association with the E3 ubiquitin ligase TRIM21. This interaction leads to the promotion of KCTD9 degradation via the ubiquitin-proteasome pathway. KCTD9, as a tumor suppressor, its low levels activated Notch signaling pathway, ultimately contributes to the CRCSC properties, the progression of CRC and liver metastasis.

Article Snippet: Antibodies for TAF1 (#12781), HES1 (#11988), CXCR4 (#64837), CXCL12 (#3740), activated Notch1 (NICD, #4380), CBP (#7389 T), P300 (#86377), TRIM21 (#92043), HA (#3724), Actin (#4967S) and His (#12698) were from CST (Danvers, MA); anti-β-Hydroxybutyryllysine (#PTM-1201RM) was from PTM BIO (Chicago, USA); antibody for KCTD9 (ab180937) were from Abcam Biotechnology (Abcam, Cambridge, UK); HEY1 (#19929-1-AP), MYC (#10828-1-AP), SNAI1 (#13099-1-AP) were from Proteintech; and anti-KCTD9 K123bhb and K129bhb rabbit polyclonal antibody generated by Proteintech using the peptide DKGVWGN-K(bhb)-QDHRGAF and D-K(bhb)-GVWGNK; and KCTD9-Kbhb peptide was synthesized by using the peptide of DK(bhb)GVWGN-K(bhb)-QDHRGAF; an antibody for FLAG (F3165) was from Sigma-Aldrich; antibodies for GAPDH (sc-25778) and GST (sc-138) were from Santa Cruz Biotechnology (Santa Cruz, CA); the secondary antibodies, anti-rabbit IgG, HRP-linked antibody (#7074) and anti-mouse IgG, HRP-linked antibody (#7076) were purchased from CST (Danvers, MA).

Techniques: Expressing, Comparison, Quantitative Proteomics, Modification, Ubiquitin Proteomics

Journal: Cancer Immunology Research

Article Title: FAM135B Deficiency Inhibits Cytotoxic T-cell Activity in Triple-Negative Breast Cancer by Blocking the IFI16-Dependent STING Pathway

doi: 10.1158/2326-6066.CIR-25-0310

Figure Lengend Snippet: FAM135B competes with TRIM21 for binding to IFI16. A and B, BT549 and MDA-MB-231 cells expressing an empty vector or FAM135B were treated with cycloheximide (CHX; 10 μmol/L) for the indicated time intervals, and cell lysates were subjected to immunoblotting using the indicated antibodies ( A ). The line graph shows the quantification of relative IFI16 levels ( B ). C, TNBC cells expressing an empty vector or FAM135B were treated with MG132 (20 μmol/L) for 6 hours. Cell lysates were immunoprecipitated using an anti-IFI16 antibody and then analyzed by immunoblotting. D, HA-tagged full-length (FL) or deletion mutants of IFI16 were co-transfected with Flag-TRIM21 into 293T cells. Cell lysates were immunoprecipitated using an anti-HA antibody and were subjected to immunoblotting analysis. E, BT549 and MDA-MB-231 cells expressing an empty vector or FAM135B were lysed, immunoprecipitated using an anti-TRIM21 antibody, and then analyzed by immunoblotting. F, Overlapping ubiquitination sites of IFI16 were identified by MS and estimated by the PhosphoSitePlus database. G, BT549 cells expressing an empty vector or TRIM21 were transfected with HA-IFI16 or its mutants (K143R or K561R). Cells were treated with MG132 (20 μmol/L) for 6 hours before harvest, and cell lysates were immunoprecipitated using an anti-HA antibody and analyzed by immunoblotting. H, TNBC cells expressing FAM135B shRNA were transfected with HA-IFI16 or its mutants (K143R or K561R). Cell lysates were analyzed by immunoblotting using the indicated antibodies. I, 293T cells were transfected with Flag-TRIM21, HA-IFI16, and Myc-Ubi or its mutants (K33R or K48R). Cells were treated with MG132 (10 μmol/L) for 6 hours before harvest, and cell lysates were immunoprecipitated using an anti-HA antibody, followed by immunoblotting analysis. IP, immunoprecipitated; p-IRF, phosphorylated IRF; p-STING, phosphorylated STING; p-TBK, phosphorylated TBK; WT, wild type.

Article Snippet: The membranes were blocked in PBS buffer supplemented with 0.1% Tween containing 5% nonfat milk for 1 hour at room temperature, followed by overnight incubation with the specified primary antibodies at 4°C: anti-FAM135B (28061-1-AP, Proteintech), anti-IFI16 (YT2274, Immunoway), anti-TRIM21 (12108-1-AP, Proteintech), anti-Flag (20543-1-AP, Proteintech), anti-HA (51064-2-AP, Proteintech), anti-MYC (16286-1-AP, Proteintech), anti–β-tubulin (66240-1-Ig, Proteintech), anti-γH2AX (YP0128, Immunoway), anti-Ubiquitin (43124, Cell Signaling Technology), anti–TANK-binding kinase 1 (67211-1-Ig, Proteintech), anti-stimulator of interferon genes (19851-1-AP, Proteintech), anti-IRF3 (11312-1-AP, Proteintech), anti–p-TBK1 (AF8190, Affinity), anti–p-STING (AF7416, Affinity), and anti–p-IRF3 (80519-2-RR, Proteintech).

Techniques: Binding Assay, Expressing, Plasmid Preparation, Western Blot, Immunoprecipitation, Transfection, Ubiquitin Proteomics, shRNA

Journal: Cell communication and signaling : CCS

Article Title: Proteomic studies of VEGFR2 in human placentas reveal protein associations with preeclampsia, diabetes, gravidity, and labor.

doi: 10.1186/s12964-024-01567-0

Figure Lengend Snippet: Fig. 3 Immunohistochemical staining of placental sections. A VEGFR2 immunostaining is strongly positive in villous endothelial cells (patient Q3). B TRIM21 is uniformly positive in villous trophoblast (patient N4). Very strong staining is seen of maternal leukocytes (arrows). C MDMX is strongly positive in the cytoplasm of HC and moderately positive in endothelial cells (patient W2). D CD163 is strongly positive in the cytoplasm of the HC (patient U1). E PICALM is strongly positive in the trophoblast (patient R4). F PICALM positivity seen in the villous endothelial cells and fetal blood leukocytes (patient R4). Magnifications in A-F were 10x, 20x and 40x

Article Snippet: VEGFR2 (55B11) no. 2479 (RRID AB_2219274) Cell Signaling (Danvers, MA), MDMX A300-287A (RRID AB_ 263407) Bethyl Laboratories (Montgomery, TX), PDC-E2 SC-365276 (RRID AB_10858873) Santa Cruz Biotechnology (Santa Cruz, CA), PICALM HPA019053 (RRID AB_1855361) Sigma-Aldrich (St. Louis, MO), OT-R ABN1735 (RRID AB_2864776) Millipore (Temecula, CA), V1aR MBS176788 (RRID AB_3068011) MyBiosource (San Diego, CA), CD163 MA5-33091 (RRID AB_2810183) InVitrogen-Thermo Fisher Scientific (Waltham, MA), TRIM21 NBP1-33548 (RRID AB_10004085) Novus (Centennial, CO), Alexa-488 (green) and Alexa-64 (red) goat anti-rabbit/mouse conjugated secondary antibodies/Life Technologies (Eugene, OR), immunogold donkey-anti-rabbit secondary antibodies 25702 (6 nm) and 25705 (10 nm) (Electron Microscopy Sciences (Hatfield, PA).

Techniques: Immunohistochemical staining, Staining, Immunostaining

Journal: Bone research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling.

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: Fig. 1 Trim21 is elevated in osteoporotic patients, and its deficiency leads to high bone mass. a Quantitative RT‒PCR analysis of Trim21 mRNA expression in bone specimens from patients with different bone mineral densities (BMDs), which were defined as normal, osteopenia, and osteoporosis. b Correlation analysis between Trim21 mRNA expression and RF-BMD and LS-BMD. RF, right femur; LS, lumbar spine. c Immunoblotting analysis of Trim21 protein expression in the lumbar vertebra of 5-month-old sham-operated or ovariectomized mice. d Schematic diagram showing the analysis of skeletal parameters of mice at different ages. e Alcian blue/Alizarin Red staining of the whole skeleton of 1-week-old Trim21+/+, Trim21+/−, and Trim21−/−littermates. f X-ray images of Trim21+/+ and Trim21−/−mice at 1 month and 6 months (left panel). Quantitative analysis of the tibia length of mice at different ages (right panel). g Representative H&E and S/O staining images of tibial sections from 1-month-old Trim21+/+ and Trim21−/−mice (left panel). Quantitative analysis of the growth plate thickness of the indicated mice (right panel). h, i Representative immunofluorescence images (h) showing the expression of Sox9+ cells (i) in growth plates of tibial sections in 1-month-old Trim21+/+ and Trim21−/−mice. j Representative micro-CT images of the proximal tibia bone of 14-week-old mice. Quantitative measurements of bone volume per tissue volume (BV/TV), trabecular thickness (Tb. Th), trabecular number (Tb. N), and trabecular separation (Tb.Sp). All bar graphs are presented as the mean ± SD. *P < 0.05; ***P < 0.001; ****P < 0.000 1; n.s. not significant by Student’s t test

Article Snippet: Establishment of global and conditional Trim21 knockout mice Heterozygous recombinant Trim21 mice (Trim21−/+) with a C57BL/ 6 genetic background were purchased from Cyagen Biosciences (Guangzhou, China).

Techniques: Expressing, Western Blot, Staining, Micro-CT

Journal: Bone research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling.

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: Fig. 2 Loss of Trim21 enhances osteoblast activity and favors bone formation. a, b Representative immunoblotting analysis (a) and quantification of Runx2, Osterix, and Trim21 in MC3T3-E1 cells (b) treated with osteogenic medium for 0, 4, and 7 days. c Quantitative RT‒PCR analysis of osteogenic biomarker genes (Osterix, Runx2, and Trim21) in OBs with osteogenic induction. d, e Alizarin Red S (upper panel) and ALP (lower panel) staining of primary osteoblasts (OBs) after induction with osteogenic medium for different times (d). The percentage of Alizarin Red S- (n ≥3) and ALP- (n ≥3) stained area (e). f Quantitative RT‒PCR detection of osteogenic biomarker genes (Runx2, Osterix, OCN, OPG, and RANKL) in OBs derived from Trim21−/−and Trim21+/+ mice upon osteogenic induction for 7 days. g, h Representative immunoblotting analysis (g) and quantification of Runx2 and Osterix in OBs (h) after osteogenic induction for 0, 4, and 7 days. i Representative micro-CT images of calvarial bone defects in 2-month-old Trim21+/+ and Trim21−/−mice after surgical induction for 1 month (left panel). Quantitative measurements of bone volume per tissue volume (BV/TV) and bone defect diameter (right panel). All bar graphs are presented as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.000 1; n.s. not significant by Student’s t test

Article Snippet: Establishment of global and conditional Trim21 knockout mice Heterozygous recombinant Trim21 mice (Trim21−/+) with a C57BL/ 6 genetic background were purchased from Cyagen Biosciences (Guangzhou, China).

Techniques: Activity Assay, Western Blot, Biomarker Discovery, Staining, Derivative Assay, Micro-CT

Journal: Bone research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling.

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: Fig. 3 Loss of Trim21 inhibits osteoclast formation and differentiation. a Representative image of histological sections of the tibia that were stained with TRAP (left panel). Bone marrow (BM) and trabecular bone (TB) are indicated in black. TRAP-stained osteoclasts (OCs) are denoted by the red arrow. OC. N/BPm (OC number per bone parameter) and OC. S/BS (OC surface per bone surface) were determined (right panel). Scale bar: 100 μm. b Quantification of F-actin ring number in BMM-derived OCs from immunofluorescence staining of Fig. S6e. c Representative immunoblot analysis and quantification of Ctsk expression in BMM-derived OCs from Trim21+/+ and Trim21−/−mice. d, e Quantitative RT‒PCR detection of OC differentiation genes (Ctsk, Nfatc1, Acp5, ATP6vod2, and Mmp9) in BMM-derived OCs from Trim21+/+

Article Snippet: Establishment of global and conditional Trim21 knockout mice Heterozygous recombinant Trim21 mice (Trim21−/+) with a C57BL/ 6 genetic background were purchased from Cyagen Biosciences (Guangzhou, China).

Techniques: Staining, Derivative Assay, Western Blot, Expressing

Journal: Bone research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling.

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: Fig. 4 YAP1/β-catenin signaling is essential for Trim21-mediated osteogenic differentiation. a Schematic diagram showing TMT-based quantitative proteomics for the identification of differentially expressed proteins (DEPs) in bone marrow mesenchymal stem cells (BMSCs) derived from Trim21+/+ and Trim21−/−mice. b Volcano plots of DEPs in BMSCs from Trim21+/+ and Trim21−/−mice. c Heatmap analysis of DEPs in BMSCs. Three replicates of each group were included, and the top 29 DEPs are shown. d KEGG enrichment analysis of the DEPs in the BMSCs. e Representative immunoblotting analysis and quantification of BCL9, AXIN1, and YAP1 in BMSCs; proteomics sample: part of the samples subjected to proteomics analysis. f Representative immunoblotting analysis and quantification of BCL9, β-catenin, YAP1, and Runx2 protein expression in BMSCs after osteogenic induction for 7 days. g The endogenous interaction between Trim21, BCL9, β-catenin, and YAP1 was evaluated using a co-IP assay. h Protein‒protein interaction of YAP1 and Trim21 in living cells. The two BiFC plasmids encoding Myc- VN155-YAP1 and HA-VC155-Trim21 along with HA-cerulean were cotransfected into HEK293T cells for 24 h. Representative images showing transfected cells (cerulean) and the interaction between YAP1 and Trim21 (Venus). Nuclei were stained with DAPI. Scale bar: 20 μm. i Immunoblotting analysis of BCL9, β-catenin, YAP1, and HA-Trim21 protein expression in HEK293T cells treated with or without MG132. j Representative images showing the expression of YAP1+ OBs derived from Trim21+/+ and Trim21−/−mice. Scale bar: 20 μm. All bar graphs are presented as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.000 1; n.s. not significant by Student’s t test

Article Snippet: Establishment of global and conditional Trim21 knockout mice Heterozygous recombinant Trim21 mice (Trim21−/+) with a C57BL/ 6 genetic background were purchased from Cyagen Biosciences (Guangzhou, China).

Techniques: Quantitative Proteomics, Derivative Assay, Western Blot, Expressing, Co-Immunoprecipitation Assay, Transfection, Staining

Journal: Bone research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling.

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: Fig. 5 Loss of Trim21 protects mice from lipopolysaccharide (LPS)-induced bone loss. a Quantitative RT‒PCR determination of IL-6, Osterix, and Runx2 mRNA expression in OBs with or without lipopolysaccharide (LPS) treatment during osteogenic induction. b Schematic diagram showing the H&E staining and micro-CT analysis of Trim21+/+ and Trim21−/−mice induced by PBS or LPS. c Representative images of H&E staining of tibia sections of 13-week-old Trim21+/+ and Trim21−/−mice induced by PBS or LPS. Bone marrow (BM) and trabecular bone (TB) are labeled with red arrows. d, e Representative micro-CT images (d) and BV/TV (e) of proximal tibia trabecular bone of 13-week-old Trim21+/+ and Trim21−/−mice induced by PBS or LPS. f The bone loss ratio after LPS treatment in global knockout mice (left panel) and conditional knockout mice (right panel). g Representative micro-CT images and BV/TV of proximal tibia trabecular bone of 13-week-old Trim21f/f and Ctsk-cre; Trim21f/f mice induced by PBS or LPS. h Representative micro-CT images of cortical bones and quantification of BV/TV (left panel) and thickness (right panel) of Trim21f/f and Ctsk-cre; Trim21f/f mice induced by either PBS or LPS. All bar graphs are presented as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; n.s. not significant by Student’s t test

Article Snippet: Establishment of global and conditional Trim21 knockout mice Heterozygous recombinant Trim21 mice (Trim21−/+) with a C57BL/ 6 genetic background were purchased from Cyagen Biosciences (Guangzhou, China).

Techniques: Expressing, Staining, Micro-CT, Labeling, Knock-Out

Journal: Bone research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling.

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: Fig. 6 Trim21 orchestrates ovariectomy (OVX)-induced bone metabolism by targeting YAP1 signaling. a Determination of fat cell density in the proximal tibia of 20-week-old Trim21+/+ and Trim21−/−mice induced by sham operation or OVX. b Representative micro-CT images and quantitative data (BV/TV) of proximal tibial bone of 20-week-old Trim21+/+ and Trim21−/−mice induced by sham operation or OVX. c, d Calcein double labeling of mineral layers of tibial trabecular bone of 5-month-old mice. e Representative images of von Kossa staining of the undecalcified proximal tibia of 5-month-old mice. Scale bar: 50 μm. f IHC staining images of the proximal tibia of 5-month-old mice using an antibody against YAP1. The YAP1-stained positive cells are denoted by the red arrow. Scale bar: 50 μm. g, h Representative images of histological sections of the tibia that were stained with TRAP in Trim21+/+ and Trim21−/−mice induced by sham operation or OVX. TRAP-stained osteoclasts (OCs) are denoted by the red arrow. OC. N/BPm (OC number per bone parameter) and OC. S/BS (OC surface per bone surface) was determined. Scale bar: 100 μm. All bar graphs are presented as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.000 1; n.s. not significant by Student’s t test

Article Snippet: Establishment of global and conditional Trim21 knockout mice Heterozygous recombinant Trim21 mice (Trim21−/+) with a C57BL/ 6 genetic background were purchased from Cyagen Biosciences (Guangzhou, China).

Techniques: Micro-CT, Labeling, Staining, Immunohistochemistry

Journal: Bone research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling.

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: Fig. 7 A schematic of Trim21 in the regulation of bone remodeling via YAP1/β-catenin signaling. Normal bone remodeling is maintained by the balance of MSC/osteoblast-mediated bone formation and osteoclast-mediated bone resorption. Trim21, by interacting with the protein complex formed by YAP1/β-catenin/BCL9, dictates the degradation of this protein complex, which in turn inactivates YAP1 and β-catenin signaling, which is essential for osteoblast differentiation. However, Trim21 is critical for maintaining the basic expression of osteoclast biomarkers, including Nfatc1 and Ctsk. Therefore, the coupling of osteoblasts with osteoclasts is attributed to dynamic changes in bone metabolism (left panel). In contrast, the loss of Trim21 causes disassociation with the YAP1/β-catenin/BCL9 complex, which then enters the nucleus for subsequent activation of osteogenic genes, including Runx2 and Osterix. In addition, the loss of Trim21 suppresses the maturation of osteoclasts. Together, these results indicate that Trim21 deficiency alleviates pathological bone loss by activating YAP1/β-catenin signaling

Article Snippet: Establishment of global and conditional Trim21 knockout mice Heterozygous recombinant Trim21 mice (Trim21−/+) with a C57BL/ 6 genetic background were purchased from Cyagen Biosciences (Guangzhou, China).

Techniques: Expressing, Activation Assay

Journal: Scientific Reports

Article Title: Leukocyte immunoglobulin-like receptor B4 regulates key signalling molecules involved in FcγRI-mediated clathrin-dependent endocytosis and phagocytosis

doi: 10.1038/srep35085

Figure Lengend Snippet: Mascot search results of mass spectrometric peptides sequencing of tyrosine phosphorylated proteins following FcγRI cross linking of THP-1 cells (n = 3).

Article Snippet: The following antibodies were used for Western blotting: biotinylated mouse α-pTyr-100 mAb (Cell Signaling, Danvers, MA, USA), mouse anti-human clathrin (Thermo Fisher Scientific, Waltham, MA, USA), mouse anti-HSP70 (Stressgen/Enzo Life Sciences, Farmingdale, NY, USA), rabbit anti-HGS (Thermo Fisher Scientific), rabbit anti-Cbl (Sigma-Aldrich), rabbit anti-FcγRs (Upstate Biotechnology Inc, Lake placid, NY, USA), rabbit anti-Syk (Cell Signaling), rabbit anti-pSyk (Tyr 525/526) (Cell Signaling), mouse anti-β-actin (Sigma-Aldrich), and mouse anti-human TRIM21 mAb (R&D System) in-house labelled with biotin using lightning-link TM biotin conjugation kit (Innova Biosciences, Babraham, Cambridge, UK), primary antibodies, and HRP-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies, and HRP-conjugated streptavidin (all from Bio-Rad, Gladesville, NSW, Australia).

Techniques: Sequencing, Control, Ubiquitin Proteomics, Binding Assay, Variant Assay, Activation Assay

Journal: Scientific Reports

Article Title: Leukocyte immunoglobulin-like receptor B4 regulates key signalling molecules involved in FcγRI-mediated clathrin-dependent endocytosis and phagocytosis

doi: 10.1038/srep35085

Figure Lengend Snippet: ( A ) Representative immunoprecipitation of THP-1 cell lysates using anti-pTyr mAb (4G10) followed by Western blotting using selected antibodies showed abundant Tyr phosphorylation of FcγR s, clatherin, HSP70, Cbl, HGS and TRIM21 in cells co-ligated with anti-FcγRI+IgG 1 control mAb validating LC-MS/MS data. Importantly, LILRB4 co-ligation with FcγRI markedly reduced Tyr phosphorylation of all proteins except for HSP70 (n = 3). ( B ) Summary of densitometry of bands from 3 independent experiments showed significant reduction of FcγRs, clatherin, Cbl, HGS and TRIM21 phosphorylation, but not HSP70, in THP-1 cells co-ligated with anti-FcγRI and anti-LILRB4 mAbs, compared to cells co-ligated with anti-FcγRI and negative control mAb (n = 3, **p < 0.01; ***p < 0.001). ( C ) Representative Western blotting of total cell lysates showed that co-ligation of FcγRI with LILRB4 did not alter the total amounts of any of the above proteins when compared to co-ligation of FcγRI+IgG 1 control, ligation of LILRB4 alone or treatment with IgG 1 control alone; the lower panel is the same membrane stripped and re-probed with anti-β actin Ab, confirming comparable protein loading. ( D ) Summary of densitometry analysis of 3 independent experiments showed no significant differences in total FcγRs, clatherin, HSP70, Cbl, HGS and TRIM21 in THP-1 cells within the 4 different treatment groups (n = 3). Full image of the Western blots is shown in . ( E ) Western blotting of cell lysates from FcγRI cross-linked cells showing increased Tyr-phosphorylated Syk that was markedly reduced upon co-ligation with LILRB4, confirming our earlier finding and validating current LC-MS/MS data (n = 1).

Article Snippet: The following antibodies were used for Western blotting: biotinylated mouse α-pTyr-100 mAb (Cell Signaling, Danvers, MA, USA), mouse anti-human clathrin (Thermo Fisher Scientific, Waltham, MA, USA), mouse anti-HSP70 (Stressgen/Enzo Life Sciences, Farmingdale, NY, USA), rabbit anti-HGS (Thermo Fisher Scientific), rabbit anti-Cbl (Sigma-Aldrich), rabbit anti-FcγRs (Upstate Biotechnology Inc, Lake placid, NY, USA), rabbit anti-Syk (Cell Signaling), rabbit anti-pSyk (Tyr 525/526) (Cell Signaling), mouse anti-β-actin (Sigma-Aldrich), and mouse anti-human TRIM21 mAb (R&D System) in-house labelled with biotin using lightning-link TM biotin conjugation kit (Innova Biosciences, Babraham, Cambridge, UK), primary antibodies, and HRP-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies, and HRP-conjugated streptavidin (all from Bio-Rad, Gladesville, NSW, Australia).

Techniques: Immunoprecipitation, Western Blot, Phospho-proteomics, Control, Liquid Chromatography with Mass Spectroscopy, Ligation, Negative Control, Membrane

Journal: Scientific Reports

Article Title: Leukocyte immunoglobulin-like receptor B4 regulates key signalling molecules involved in FcγRI-mediated clathrin-dependent endocytosis and phagocytosis

doi: 10.1038/srep35085

Figure Lengend Snippet: Cross-linking of FcγRI by immune-complexes causes Tyr phosphorylation of the ITAMs of its common γ chain and binding of pSyk transduces activating signals. This simultaneously initiates phosphorylation of clatherin that causes lateral diffusion of receptor-ligand complexes to clathrin-coated pits, membrane invagination and generation of clathrin-coated vesicles, and/or initiates phosphorylation of Cbl that may directly ubiquitinate the receptor. Phosphorylated Cbl triggers phosphorylation of HSP70 that facilitates un-coating of the vesicles, a precondition for vesicles to fuse with early endosomes and release ligands. The released receptors are transported to either the late endosome and/or lysosome for proteosomal and/or lysosomal degradation or are recycled to the cell surface. The immune complexes in the endosome are either directly degraded by Cbl, or delivered to the lysosome by phosphorylated HGS-STAM 1/2 complex for final degradation. During transfer, immune complexes that escape the endosome are recognised by phosphorylated TRIM21 for proteasomal degradation. Co-ligation of FcγRI with LILRB4 may recruit phosphatases such as SHP-1 to its ITIMs that subsequently dephosphorylate (deactivate) the key molecules including clathrin (1), FcγRI and Syk (2), Cbl (3), HGS and STAM 1/2 (4) and TRIM21(5). These effects may reduce cellular activation and/or suppress receptor/ligand endocytosis. *New Tyr phosphorylated and dephosphorylated proteins identified in this study.

Article Snippet: The following antibodies were used for Western blotting: biotinylated mouse α-pTyr-100 mAb (Cell Signaling, Danvers, MA, USA), mouse anti-human clathrin (Thermo Fisher Scientific, Waltham, MA, USA), mouse anti-HSP70 (Stressgen/Enzo Life Sciences, Farmingdale, NY, USA), rabbit anti-HGS (Thermo Fisher Scientific), rabbit anti-Cbl (Sigma-Aldrich), rabbit anti-FcγRs (Upstate Biotechnology Inc, Lake placid, NY, USA), rabbit anti-Syk (Cell Signaling), rabbit anti-pSyk (Tyr 525/526) (Cell Signaling), mouse anti-β-actin (Sigma-Aldrich), and mouse anti-human TRIM21 mAb (R&D System) in-house labelled with biotin using lightning-link TM biotin conjugation kit (Innova Biosciences, Babraham, Cambridge, UK), primary antibodies, and HRP-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies, and HRP-conjugated streptavidin (all from Bio-Rad, Gladesville, NSW, Australia).

Techniques: Phospho-proteomics, Binding Assay, Diffusion-based Assay, Membrane, Ligation, Activation Assay

Journal: Experimental & Molecular Medicine

Article Title: The ubiquitin E3 ligase TRIM21 suppresses type I interferon signaling via STING degradation and ameliorates systemic autoimmunity

doi: 10.1038/s12276-025-01490-5

Figure Lengend Snippet: a Western blots and densitometry analysis of Trim21 in splenocytes from female MRL/ lpr mice (9 weeks, n = 4; 17 weeks, n = 4; 23 weeks, n = 5). b Spleen CD19 + B cells from 8-week-old female MRL/ lpr mice were transfected with Trim21 siRNA and stimulated with CD40 ligand and IL-4 for 4 days. Levels of total IgG and IgG2a in the culture supernatant were determined using ELISA. c – k Eight-week-old female MRL/ lpr mice were injected with mock ( n = 4) or Trim21 overexpression ( n = 4) vector for 8 weeks: weights and lengths of spleens ( c ) and percentage of spleen weight relative to body weight ( d ); representative photomicrographs of PAS-stained kidney tissues of MRL/ lpr mice ( e ) with histopathologic analysis ( f ) (in e , glomerular and tubular images (top) and vascular images (bottom) are shown; scale bar, 100 μm); serum levels of anti-dsDNA antibodies (total IgG) in MRL/ lpr mice measured using ELISA ( g ); flow cytometric analysis of double-negative T cells in peripheral blood of 16-week-old MRL/ lpr mice (live T cells were defined as Fixable Viability Dye − CD90.2 + cells) ( h ); flow cytometric analysis of regulatory B cells ( i ) and pDCs or IFNα-producing pDCs ( j ) in splenocytes of 16-week-old MRL/ lpr mice; flow cytometric analysis of GC B cells, plasma B cells and IL-10- or IL-17-producing B cells in peripheral blood of 16-week-old MRL/ lpr mice ( k ). All data are shown as mean ± s.e.m. Statistical analyses were performed using one-way ANOVA ( a ), two-tailed paired t -test ( b – d , f and h – k ) or two-way ANOVA ( g ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The interaction between TRIM21 and STING was validated using antibodies against TRIM21 (15-060, ProSci) and STING (sc-518172, Santa Cruz Biotechnology) in human PBMCs and mouse splenocytes.

Techniques: Western Blot, Transfection, Enzyme-linked Immunosorbent Assay, Injection, Over Expression, Plasmid Preparation, Staining, Clinical Proteomics, Two Tailed Test

Journal: Experimental & Molecular Medicine

Article Title: The ubiquitin E3 ligase TRIM21 suppresses type I interferon signaling via STING degradation and ameliorates systemic autoimmunity

doi: 10.1038/s12276-025-01490-5

Figure Lengend Snippet: a , d Flow cytometric analysis of B cells ( a ) and pDCs ( d ) in splenocytes from WT B6 ( n = 10) and Trim21 −/− ( n = 9) mice. In a , total B220 + cells, GC B cells, plasma B cells and regulatory B cells were analyzed. In d , pDCs and IFNα-producing pDCs were analyzed. b , c Splenic B220 + B cells were stimulated with CD40 ligand, anti-IgM and IL-4. mRNA levels of Blimp1 , Xbp1 , Bcl6 and Pax5 in the cells were determined using qPCR ( b ), and levels of total IgG, IgG1, IgG2a and IgG3 in the culture supernatant were determined using ELISA ( c ). All data are shown as mean ± s.e.m. Statistical analyses were performed using two-tailed paired t -test ( a , b and d ) or two-way ANOVA ( c ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The interaction between TRIM21 and STING was validated using antibodies against TRIM21 (15-060, ProSci) and STING (sc-518172, Santa Cruz Biotechnology) in human PBMCs and mouse splenocytes.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Experimental & Molecular Medicine

Article Title: The ubiquitin E3 ligase TRIM21 suppresses type I interferon signaling via STING degradation and ameliorates systemic autoimmunity

doi: 10.1038/s12276-025-01490-5

Figure Lengend Snippet: Eight-week-old female WT B6 and Trim21 −/− mice were treated with vehicle (acetone alone, n = 4) or R848 ( n = 4) for 31 days. a – c Representative photomicrographs of PAS-stained kidney tissues of mice with histopathologic analyses. Glomerular and tubular images ( a ) and vascular images ( b ) are shown (scale bar, 100 μm). d Flow cytometric analysis of GC B cells, plasma B cells and regulatory B cells in splenocytes from mice. e , f Flow cytometric analysis of the STING pathway in splenocytes from mice. The bar graphs represent the relative Mean Fluorescence Intensity (MFI) values analyzed in CD19 + B cells ( e ) or Siglec-H + PDCA1 + Freq (%) of CD11c + DCs ( f ). g , h Confocal images of spleen sections stained for CD19 (green) and Cxcl10 (red) ( g ) or PDCA1 (red) and IFNα (green) ( h ) (scale bar, 20 μm). Cell counts are shown on the right. All data are shown as mean ± s.e.m. Statistical analyses were performed using one-way ANOVA ( c and d ) or two-tailed paired t -test ( e – h ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The interaction between TRIM21 and STING was validated using antibodies against TRIM21 (15-060, ProSci) and STING (sc-518172, Santa Cruz Biotechnology) in human PBMCs and mouse splenocytes.

Techniques: Staining, Clinical Proteomics, Fluorescence, Two Tailed Test

Journal: Experimental & Molecular Medicine

Article Title: The ubiquitin E3 ligase TRIM21 suppresses type I interferon signaling via STING degradation and ameliorates systemic autoimmunity

doi: 10.1038/s12276-025-01490-5

Figure Lengend Snippet: a Representative images of 7-month-old WT ( Trim21 +/+ ) and Trim21 −/− B6. lpr mice with their spleen (scale bar, 1 cm). b The spleen weights of 7-month-old Trim21 +/+ ( n = 5) and Trim21 −/− ( n = 13) B6. lpr mice. c ACR measured from urine of 10-month-old Trim21 +/+ ( n = 4) and Trim21 −/− ( n = 4) B6. lpr mice. d , e Representative photomicrographs of PAS-stained kidney tissues of 7-month-old Trim21 +/+ ( n = 5) and Trim21 −/− ( n = 13) B6. lpr mice with histopathologic analyses. Glomerular and tubular image (top) and vascular image (bottom) are shown ( d ; scale bar, 100 μm). f IgG2a measured from serum of Trim21 +/+ and Trim21 −/− B6. lpr mice using ELISA. g Flow cytometric analysis of GC B cells, plasma B cells, regulatory B cells, pDCs and IFNα-producing pDCs in peripheral blood of 5-month-old Trim21 +/+ ( n = 5) and Trim21 −/− ( n = 11) B6. lpr mice. h Flow cytometric analysis of GC B cells, plasma B cells, regulatory B cells, pDCs and IFNα-producing pDCs in splenocytes of 7-month-old Trim21 +/+ ( n = 5) and Trim21 −/− ( n = 15) B6. lpr mice. i , j Flow cytometric analysis of the STING pathway in splenocytes. The bar graphs represent the relative MFI values analyzed in CD19 + B cells ( i ) or Siglec-H + PDCA1 + Freq (%) of CD11c + DCs ( j ). All data are shown as mean ± s.e.m. Statistical analyses were performed using two-tailed paired t -test ( b , c , e and g – j ) or two-way ANOVA ( f ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ACR albumin/creatinine ratio.

Article Snippet: The interaction between TRIM21 and STING was validated using antibodies against TRIM21 (15-060, ProSci) and STING (sc-518172, Santa Cruz Biotechnology) in human PBMCs and mouse splenocytes.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Two Tailed Test

Journal: Experimental & Molecular Medicine

Article Title: The ubiquitin E3 ligase TRIM21 suppresses type I interferon signaling via STING degradation and ameliorates systemic autoimmunity

doi: 10.1038/s12276-025-01490-5

Figure Lengend Snippet: a HEK293 cells were immunoprecipitated with an anti-STING antibody or normal IgG, and immunoblotted using anti-TRIM21 and anti-STING antibodies. b NIH3T3 cells were transfected with mock or mouse Trim21 overexpression vector (3 μg) with MG132 (5 μM) treatment. The cells were immunoprecipitated with anti-Sting antibodies or normal IgG and immunoblotted using anti-ubiquitin, anti-Sting and anti-Trim21 antibodies. c In vitro ubiquitination assay using recombinant TRIM21 and STING proteins. The reaction mixture was incubated at 37 °C for 1 h. Proteins were separated by SDS–PAGE and immunoblotted using anti-ubiquitin, anti-TRIM21 and anti-STING antibodies. d PLA to detect protein interactions between mCherry and Sting in NIH3T3 cells overexpressing pmCherry-C1- Trim21 , pmCherry-C1- Trim21ΔRING-Box and pmCherry-C1- Trim21ΔPRYSPRY . Nuclei were stained with DAPI (scale bar, 20 μm). e NIH3T3 cells were transfected with pmCherry-C1 mock or Trim21 overexpression vectors and treated with MG132 (5 μM). Cells were immunoprecipitated with anti-Sting antibodies and immunoblotted with anti-ubiquitin, anti-Sting and anti-mCherry antibodies. f Western blots and densitometry analysis of cells for Sting and Trim21. NIH3T3 cells were treated with MG132 (5 μM) or NH 4 Cl (10 mM). g Western blots and densitometry analysis of splenocytes from WT B6 and Trim21 −/− mice for Sting and Trim21. h Splenocytes from WT B6 and Trim21 −/− mice were stimulated with H-151 for 2 days. IFNα-producing pDCs in the cells were analyzed using flow cytometry. All data are shown as mean ± s.e.m. Statistical analyses were performed using one-way ANOVA ( d , f and h ) or two-tailed paired t -test ( g ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The interaction between TRIM21 and STING was validated using antibodies against TRIM21 (15-060, ProSci) and STING (sc-518172, Santa Cruz Biotechnology) in human PBMCs and mouse splenocytes.

Techniques: Immunoprecipitation, Transfection, Over Expression, Plasmid Preparation, Ubiquitin Proteomics, In Vitro, Recombinant, Incubation, SDS Page, Staining, Western Blot, Flow Cytometry, Two Tailed Test

Journal: Experimental & Molecular Medicine

Article Title: The ubiquitin E3 ligase TRIM21 suppresses type I interferon signaling via STING degradation and ameliorates systemic autoimmunity

doi: 10.1038/s12276-025-01490-5

Figure Lengend Snippet: a HEK293 cells were transfected with mock or human TRIM21 overexpression vector (3 μg) with MG132 (5 μM) treatment. The cells were immunoprecipitated with anti-STING antibodies or normal IgG, and immunoblotted using anti-ubiquitin, anti-STING and anti-TRIM21 antibodies. b Western blots and densitometry analysis of the cells for STING and TRIM21. HEK293 cells were treated with MG132 (1 μM) or NH 4 Cl (1 μM). c , d Western blots ( c ) and densitometry analysis ( d ) of PBMCs from HCs ( n = 7) and patients with SLE ( n = 7) for TRIM21 and STING. e qPCR of PBMCs from HCs ( n = 26) and patients with SLE ( n = 58) for TRIM21 . f Confocal images of PBMCs from HCs ( n = 8) and patients with SLE ( n = 8) for TRIM21 (green), STING (red) and IFNα (white) with DAPI (blue) (scale bar, 20 μm). g , h Stained cell counts of TRIM21 + cells, STING + cells, and IFNα + cells ( g ) or TRIM21 + STING − cells, TRIM21 − STING + cells, and TRIM21 − IFNα + cells ( h ). i , j Correlation between stained cell counts in patients with SLE. Correlation of TRIM21 + cell counts and STING + cell counts ( i ) or TRIM21 + cell counts and IFNα + cell counts ( j ). k , m Flow cytometric analysis of TRIM21 and STING in PBMCs from HCs ( n = 4) and patients with SLE ( n = 4). The bar graphs represent the MFI values analyzed in CD19 + B cells ( k ) and CD11c + DCs ( m ). l qPCR of PBMCs from HCs ( n = 13) and patients with SLE ( n = 26) for CXCL10 . n qPCR of PBMCs from HCs ( n = 13) and patients with SLE ( n = 58) for IFNA2 . All data are shown as mean ± s.e.m. Statistical analyses were performed using one-way ANOVA ( b ), two-tailed paired t -test ( d , e , g , h and k – n ) or Pearson’s correlation analysis ( i and j ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The interaction between TRIM21 and STING was validated using antibodies against TRIM21 (15-060, ProSci) and STING (sc-518172, Santa Cruz Biotechnology) in human PBMCs and mouse splenocytes.

Techniques: Transfection, Over Expression, Plasmid Preparation, Immunoprecipitation, Ubiquitin Proteomics, Western Blot, Staining, Two Tailed Test

Journal: Experimental & Molecular Medicine

Article Title: The ubiquitin E3 ligase TRIM21 suppresses type I interferon signaling via STING degradation and ameliorates systemic autoimmunity

doi: 10.1038/s12276-025-01490-5

Figure Lengend Snippet: a , b Correlations between TRIM21 mRNA ( a ) and TRIM21 protein ( b ) expression levels in PBMCs and disease activity-related clinical parameters, including white blood cell (WBC) counts, complement (C3 and C4) levels, anti-DNA antibody levels and SLE disease activity index (SLEDAI) in patients with SLE ( n = 21). TRIM21 mRNA and TRIM21 protein expression were measured by qPCR and western blots, respectively. c , d Correlations between STING1 mRNA expression levels in PBMCs and IFNA2 mRNA expression levels ( c ) and anti-DNA antibody levels ( d ) in patients with SLE ( n = 31). STING1 and IFNA2 mRNA expression were measured by qPCR. e Correlations between activated STING (MFI values of p-STING relative to total STING) and TRIM21 mRNA expression levels in PBMCs of patients with SLE ( n = 38). The expression of STING and p-STING was measured by flow cytometry. f , g mRNA expression levels of TRIM21 ( f ) and IFNA2 ( g ) in PBMCs from anti-TRIM21 antibody-positive ( n = 6) and antibody-negative ( n = 14) patients with SLE measured by qPCR. All data are shown as mean ± s.e.m. Statistical analyses were performed using Pearson’s correlation analysis ( a – e ) or two-tailed paired t -test ( f and g ). * P < 0.05.

Article Snippet: The interaction between TRIM21 and STING was validated using antibodies against TRIM21 (15-060, ProSci) and STING (sc-518172, Santa Cruz Biotechnology) in human PBMCs and mouse splenocytes.

Techniques: Expressing, Activity Assay, Western Blot, Flow Cytometry, Two Tailed Test