Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: The Ubiquitin E3 Ligase TRIM21 Promotes Hepatocarcinogenesis by Suppressing the p62-Keap1-Nrf2 Antioxidant Pathway

doi: 10.1016/j.jcmgh.2021.01.007

Figure Lengend Snippet: Increased TRIM21 expression is associated with HCC. ( A ) In the Gene Expression Omnibus Series, 6 studies showed elevated TRIM21 expression in HCC compared with normal liver tissues. ( B ) TCGA shows increased TRIM21 TPM in liver cancer comparing with normal tissues. ( C )TRIM21 high expression indicates poor prognosis with decreasing overall survival according to TCGA data analysis, while the disease-free survival is trending lower yet not significant. ( D ) Liver tissue sections were stained for TRIM21 by immunohistochemistry (IHC). Representative TRIM21 staining on normal liver tissues from peripheral hepatic hemangioma (left panel; n = 20), on tumor tissues from HCC (middle panel; n = 49), and on paracancerous tissues 2 cm away from the HCC (right panel; n = 29). ( E ) TRIM21 expression is increased in HCC tissues compared with the paracancerous tissues and normal liver tissues. Relative intensity of staining in each section was assessed blindly and assigned a value ranging from 0 to 4 according to intensity of staining: score 0, negative staining, no positive cells; score 1, weak staining, the ratio of positive cells to total cells ≤25%; score 2, moderately positive, the ratio of positive cells to total cells ≤50%; score 3, moderately positive, the ratio of positive cells to total cells ≤75%; score 4, strongly positive, the ratio of positive cells to total cells>75%. Low expression defines the score from 0 to 2 and high expression is the score 3–4. ( F ) Receiver-operating characteristic (ROC) curves of the ability of TRIM21 or AFP high expression to the diagnosis of HCC. ( G ) TRIM21 was silenced in HCT116 and MHHC-97L human cells, or reconstituted with vector or HA-hTRIM21 in TRIM21 –/– MEFs. Cells were lysed in RIPA buffer (1% SDS) and subjected to WB. The TRIM21 antibody obtained from Bioss (bs-0635R) or Proteintech (12018-1-AP) was used to detect human TRIM21. Note that the Bioss antibody fails to recognize human TRIM21, and neither antibody recognizes murine TRIM21. ( H ) TRIM21 antibody (Bioss bs-0635R) was used for IHC on HCC tumor tissues and paracancerous tissues collected 2 cm away from the HCC. Note that the Bioss antibody fails to detect difference in IHC staining in paracancerous and HCC tissues. ∗ P < .05. ∗∗ P < .01. ∗∗∗ P < .001.

Article Snippet: The TRIM21 antibody obtained from Bioss (bs-0635R) or Proteintech (12018-1-AP) was used to detect human TRIM21.

Techniques: Expressing, Staining, Immunohistochemistry, Negative Staining, Plasmid Preparation

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: The Ubiquitin E3 Ligase TRIM21 Promotes Hepatocarcinogenesis by Suppressing the p62-Keap1-Nrf2 Antioxidant Pathway

doi: 10.1016/j.jcmgh.2021.01.007

Figure Lengend Snippet: TRIM21 ablation leads to decreased HCC induced by DEN/PB. ( A ) TRIM21 +/+ (n = 8), TRIM21 +/– (n = 15), and TRIM21 –/– (n = 14) 14-day-old male mice were intraperitoneal injected with DEN (5 mg/kg), then fed with 0.05% PB in drinking water 7 days later. Livers from DEN/PB-treated mice were collected after 10 months. Representative images are shown. ( B ) Liver weight and body weight were measured 10 months after DEN/PB treatment. Ratio of liver weight to body 8 was plotted. ( C ) Total tumor number was counted and plotted. Each dot represents 1 mouse. ( D ) Tumors with >0.3 cm diameter was counted and plotted. ( E ) Liver tissue sections were processed for hematoxylin and eosin staining. TRIM21 +/+ (left panel), TRIM21 +/– (middle panel), and TRIM21 –/– (right panel) liver tissue showed solid type hepatocellular carcinoma, with poorly differentiated (including tumor giant cell), moderately differentiated, and well differentiated, respectively. ( F ) Liver tissues sections were stained for HSP70 and PCNA. PCNA-positive cells were counted and the means of 3 randomly selected fields are shown. (G) Liver tissue sections were processed for Sirius Red staining and α-SMA/DAPI staining by immunofluorescence. Representative images are shown. ∗ P < .05. ∗∗ P < .01. ∗∗∗ P < .001. IOD, integrated optical density.

Article Snippet: The TRIM21 antibody obtained from Bioss (bs-0635R) or Proteintech (12018-1-AP) was used to detect human TRIM21.

Techniques: Injection, Staining, Immunofluorescence

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: The Ubiquitin E3 Ligase TRIM21 Promotes Hepatocarcinogenesis by Suppressing the p62-Keap1-Nrf2 Antioxidant Pathway

doi: 10.1016/j.jcmgh.2021.01.007

Figure Lengend Snippet: TRIM21 –/– livers are protected from DEN-induced liver damage. ( A , B ) Eight-week-old TRIM21 +/+ and TRIM21 –/– male mice were treated with 200-mg/kg DEN via intraperitoneal injection. Cardiac blood was collected. ( A ) Alanine aminotransferase and ( B ) aspartate aminotransferase were measured before and after 12, 24, and 48 hours treatment. ( C ) Liver tissues (control and 48 hours DEN treatment) were lysed in RIPA buffer and subjected to WB for enzymes related to DEN metabolism. (D) Hematoxylin and eosin staining of formalin-fixed, paraffin-embedded liver sections. ( E ) F-4/80 staining indicates more profound monocyte infiltration in WT livers upon DEN treatment. (F) Oil Red O (ORO) staining of frozen liver sections with hematoxylin counterstaining. Red indicates lipid, purple indicates nuclei. Representative images are shown. ( G ) Quantitation of the area occupied by lipid inclusions within the frozen liver sections after staining of sections with ORO. Data are presented as mean ± SEM of 3 replicates. ∗∗ P < .01. ∗∗∗ P < .001.

Article Snippet: The TRIM21 antibody obtained from Bioss (bs-0635R) or Proteintech (12018-1-AP) was used to detect human TRIM21.

Techniques: Injection, Staining, Formalin-fixed Paraffin-Embedded, Quantitation Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: The Ubiquitin E3 Ligase TRIM21 Promotes Hepatocarcinogenesis by Suppressing the p62-Keap1-Nrf2 Antioxidant Pathway

doi: 10.1016/j.jcmgh.2021.01.007

Figure Lengend Snippet: TRIM21 –/– livers are protected from DEN-induced liver damage. ( A ) Eight-week-old TRIM21 +/+ and TRIM21 –/– mice were treated with 200-mg/kg DEN via intraperitoneal injection. Livers were collected 48 hours later and processed for IHC analysis. Mean integrated optical density was determined for p62, Keap1, and cleaved caspase-3 (C-C3) because they are predominantly localized in the cytosol. Cells with positive nuclear staining of 8-oxo-dG and γH2A.X staining were counted and indicated as the percentage to total cells. Representative images are shown. Data are presented as mean ± SEM of 3 replicates. ( B ) Liver tissues were lysed in RIPA buffer and subjected to WB for indicated proteins. ∗∗ P < .01. ∗∗∗ P < .001.

Article Snippet: The TRIM21 antibody obtained from Bioss (bs-0635R) or Proteintech (12018-1-AP) was used to detect human TRIM21.

Techniques: Injection, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: The Ubiquitin E3 Ligase TRIM21 Promotes Hepatocarcinogenesis by Suppressing the p62-Keap1-Nrf2 Antioxidant Pathway

doi: 10.1016/j.jcmgh.2021.01.007

Figure Lengend Snippet: DEN-induced hepatocyte proliferation is suppressed in TRIM21 –/– liver. ( A ) Eight-week-old TRIM21 +/+ and TRIM21 –/– male mice were treated with PBS or 200-mg/kg DEN by intraperitoneal injection. Liver tissues were collected after 48 hours for RNA-seq. Hierarchical clustering and heatmap illustration of differentially expressed genes under indicated conditions. Color key indicates Z-scores after normalization. ( B ) Venn diagram showing overlap of genes differentially expressed in PBS-treated vs DEN-treated TRIM21 +/+ and TRIM21 –/– mice. Analysis was restricted to genes, which showed a 4-fold difference and yielded P < .05. ( C ) Dot plots showing the enriched terms from Gene Ontology analysis using differentially expressed genes identified from DEN vs PBS treatment in TRIM21 WT mice (the 577 unique genes altered by >4-fold in TRIM21-WT mice as shown in B ). Note that genes involved in redox regulation, chemical carcinogenesis, and retinol metabolism are identified. ( D ) Heatmap shows that DEN-induced expressions of hepatic stellate cell and Kupffer cell markers were markedly decreased in TRIM21 KO livers, while no obvious difference was detected in hepatocyte markers. ( E ) Quantitative reverse-transcription PCR for the DEN-treated TRIM21 WT and KO liver samples, showing decreased antioxidant response and increased proliferation signals DEN-treated WT mice (n = 3 per group). ( F ) IHC analysis of PCNA expression in livers of PBS- and DEN-treated TRIM21 WT and KO mice. PCNA-nuclear positive cells were counted and the ratio of positive cells was calculated. Representative images are shown. Data are presented as means ± SEM of 3 mice in each group. ∗∗∗ P < .001. FPKM, fragments per kilobase of transcript per million; n.s., nonsignificant.

Article Snippet: The TRIM21 antibody obtained from Bioss (bs-0635R) or Proteintech (12018-1-AP) was used to detect human TRIM21.

Techniques: Injection, RNA Sequencing Assay, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: The Ubiquitin E3 Ligase TRIM21 Promotes Hepatocarcinogenesis by Suppressing the p62-Keap1-Nrf2 Antioxidant Pathway

doi: 10.1016/j.jcmgh.2021.01.007

Figure Lengend Snippet: TRIM21 silencing protects cells from DEN-induced oxidative damage. TRIM21 –/– MEFs reconstituted with vector, HA-TRIM21 WT, HA-TRIM21 LD, and HA-TRIM21 W381/383A mutant. ( A ) Cells were lysed in RIPA buffer with 1% SDS and subjected to WB. ( B–E ) Cells were treated with DEN (20 mM) for 6 hours. ( B ) Cells were harvested and probed for indicated proteins by WB. ( C ) Cells were stained with H2DCFDA and analyzed by flow cytometry. Quantification of relative H2DCFDA intensity (geometry mean of 3 repeats) is shown on the right. ∗∗∗ P < .001. ( D ) Cells were subjected to IF with Keap1 and p62 antibodies and observed under deconvolution microscope. Cells with p62/Keap1 aggregates were counted blindly. Data shown are the averages plus SD of at least 3 countings with over 200 cells. ∗∗∗ P < .001. ( E ) Cells were separated into Triton X-100 (1%) soluble and insoluble fractions, and 30-μg soluble proteins and corresponding volume of insoluble proteins were used for WB. The numbers below Western blot panels indicate the ratios of absolute levels of insoluble and soluble proteins, respectively.

Article Snippet: The TRIM21 antibody obtained from Bioss (bs-0635R) or Proteintech (12018-1-AP) was used to detect human TRIM21.

Techniques: Plasmid Preparation, Mutagenesis, Staining, Flow Cytometry, Microscopy, Western Blot

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: The Ubiquitin E3 Ligase TRIM21 Promotes Hepatocarcinogenesis by Suppressing the p62-Keap1-Nrf2 Antioxidant Pathway

doi: 10.1016/j.jcmgh.2021.01.007

Figure Lengend Snippet: Silencing of TRIM21 protects cells from DEN-induced genotoxic damage and cell death. ( A ) SMMC-7721 cells were treated with DEN (50 mM) alone or together with N-acetylcysteine (5 mM) for indicated time. Cell viability was analyzed by PI exclusion. ∗∗∗ P < .001. ( B ) TRIM21 was silenced in SMMC-7721 cells using 2 independent short hairpin RNAs. Cells was lysed in RIPA buffer with 1% SDS and subjected to WB. ( C ) SMMC-7721 with nontargeted control, shTRIM21 #1 and shTRIM21 #2 were treated with DEN (50 mM) for indicated periods of time. Cell viability was determined by trypan blue exclusion. Shown are the mean plus SD of 3 countings. ∗ P < .05. ( D ) SMMC-7721 with nontargeted control, shTRIM21 #1, and shTRIM21 #2 were treated with DEN (50 mM) for 6 hours. Cells were lysed in immunoprecipitation lysis buffer containing 1% Triton X-100. The insoluble fraction was dissolved in RIPA buffer containing 1% SDS. Both Triton X-100 soluble and insoluble fractions were subjected to WB, and 30-μg soluble proteins and corresponding volume of insoluble proteins were used for WB. The numbers below Western blot panels indicate the ratios of absolute levels of insoluble and soluble proteins, respectively. ( E ) SMMC-7721 cells were treated with DEN (50 mM) for 6 hours. Cells were separated into cytoplasmic and nuclear fractions, and 20-μg cytosolic proteins and corresponding volume of nuclear fractions were used for WB. ( F ) SMMC-7721 cells were treated with DEN (50 mM) for 6 hours. Cells was lysed in RIPA buffer (1% SDS) and subjected to WB.

Article Snippet: The TRIM21 antibody obtained from Bioss (bs-0635R) or Proteintech (12018-1-AP) was used to detect human TRIM21.

Techniques: Immunoprecipitation, Lysis, Western Blot

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: JCI insight

Article Title: Alternative exon usage in TRIM21 determines the antigenicity of Ro52/TRIM21 in systemic lupus erythematosus.

doi: 10.1172/jci.insight.163795

Figure Lengend Snippet: Figure 2. Neutrophils express splicing variants of TRIM21/Ro52. (A) Schematic representation of the transcripts corresponding to TRIM21 isoforms found in a publicly available RNA-Seq data set (GSE149050) from classical monocytes (cMo), neutrophils (PMN), and T cells from patients with SLE (n = 24) and healthy controls (HC, n = 12) using the ‘new tuxedo’ pipeline. Each solid block represents an exon. (B–D) Differential expression analyses of TRIM21α (B), TRIM21β (C), and TRIM21γ (D) between HC and SLE according to cell type. Pairwise comparisons between HC and SLE were done using Wilcoxon’s test. *P < 0.05, **P < 0.01.

Article Snippet: Mouse monoclonal anti–human IFIT3 (H00003437-B01) was purchased from Abnova, mouse monoclonal anti–human Ro52 (clone D-12) was purchased from Santa Cruz Biotechnology Inc., rabbit anti–N-terminal TRIM21 polyclonal antibody was purchased from Origene (TA335782), rabbit anti– human TRIM21 polyclonal antibody was purchased from Proteintech (121081-1-AP), mouse anti–human TRIM21 monoclonal antibody was purchased from Proteintech (671361-1-Ig), rabbit anti–C-terminal TRIM21 polyclonal antibody was purchased from MilliporeSigma (AV381248), mouse anti–human β-actin was purchased from MilliporeSigma (A5316), and mouse anti–human histone H3 was purchased from Cell Signaling Technology (96C10).

Techniques: RNA Sequencing, Blocking Assay, Quantitative Proteomics

Journal: JCI insight

Article Title: Alternative exon usage in TRIM21 determines the antigenicity of Ro52/TRIM21 in systemic lupus erythematosus.

doi: 10.1172/jci.insight.163795

Figure Lengend Snippet: Figure 3. Autoantibodies against Ro52 target a sequence encoded by exon 4 in TRIM21. (A–C) Schematic representation of exon usage and structural domains in Ro52 isoforms. (D–I) SLE sera (D) and commercial anti-Ro52 antibodies (E–I) from Figure 1, C–H, were used to immunoblot cell lysates from HEK293 cells transfected with mock (empty vector [EV]) or plasmids expressing Ro52α, Ro52β, or Ro52γ. The arrows denote the Ro52 isoforms. Detection of Ro52α, Ro52β, and Ro52γ by the N-terminal anti-Ro52 antibody in H is also shown as a loading control. RING, really interesting new gene; BB, B-box domain; CC, coiled-coil; LZ, leucine zipper.

Article Snippet: Mouse monoclonal anti–human IFIT3 (H00003437-B01) was purchased from Abnova, mouse monoclonal anti–human Ro52 (clone D-12) was purchased from Santa Cruz Biotechnology Inc., rabbit anti–N-terminal TRIM21 polyclonal antibody was purchased from Origene (TA335782), rabbit anti– human TRIM21 polyclonal antibody was purchased from Proteintech (121081-1-AP), mouse anti–human TRIM21 monoclonal antibody was purchased from Proteintech (671361-1-Ig), rabbit anti–C-terminal TRIM21 polyclonal antibody was purchased from MilliporeSigma (AV381248), mouse anti–human β-actin was purchased from MilliporeSigma (A5316), and mouse anti–human histone H3 was purchased from Cell Signaling Technology (96C10).

Techniques: Sequencing, Western Blot, Transfection, Plasmid Preparation, Expressing, Control

Journal: JCI insight

Article Title: Alternative exon usage in TRIM21 determines the antigenicity of Ro52/TRIM21 in systemic lupus erythematosus.

doi: 10.1172/jci.insight.163795

Figure Lengend Snippet: Figure 5. Patients with SLE have autoantibodies targeting distinct Ro52 isoforms. (A) Schematic representation showing the regions encoded by TRIM21 exon 4, exons 4 and 5, exons 3 and 4, and exons 3–5 in Ro52. (B) Recombinant proteins containing the sequence encoded by TRIM21 exon 4, exons 4 and 5, exons 3 and 4, and exons 3–5 were detected by immunoblotting using SLE sera positive for anti-Ro52Ex4 antibodies. Representative data from 6 sera are shown. (C and D) Levels of antibodies to Ro52Ex4 (C) and Ro52γCT (D) in sera from the SPARE cohort (SLE) and healthy controls (HC). Comparisons were done using Student’s t test. (E) Venn diagram depicting the anti-Ro52 antibody intersections (overlap) in 128 of 191 patients with SLE positive for anti-Ro52Ex4, anti-Ro52γCT, and/or anti-Ro52‘classic’ antibodies. (F and G) Recombinant Ro52α, Ro52β, and Ro52γ and the sequence encoded by Ro52 exons 3 and 4 were used to analyze by immunoblotting SLE sera from the none overlapping anti-Ro52‘classic’ antibodies (n = 9) (representative data from 5 sera are shown) (F) and anti-Ro52Ex4 serum (G). ****P < 0.0001.

Article Snippet: Mouse monoclonal anti–human IFIT3 (H00003437-B01) was purchased from Abnova, mouse monoclonal anti–human Ro52 (clone D-12) was purchased from Santa Cruz Biotechnology Inc., rabbit anti–N-terminal TRIM21 polyclonal antibody was purchased from Origene (TA335782), rabbit anti– human TRIM21 polyclonal antibody was purchased from Proteintech (121081-1-AP), mouse anti–human TRIM21 monoclonal antibody was purchased from Proteintech (671361-1-Ig), rabbit anti–C-terminal TRIM21 polyclonal antibody was purchased from MilliporeSigma (AV381248), mouse anti–human β-actin was purchased from MilliporeSigma (A5316), and mouse anti–human histone H3 was purchased from Cell Signaling Technology (96C10).

Techniques: Recombinant, Sequencing, Western Blot

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

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