Journal: Journal of neuroinflammation

Article Title: Regulatory role of TRIM21 in the type-I interferon pathway in Japanese encephalitis virus-infected human microglial cells.

doi: 10.1186/1742-2094-11-24

Figure Lengend Snippet: Figure 1 TRIM21 attenuates the JEV mediated upregulation of the p-IRF3 level and IFN-β level in human microglial cells. (A) PCR amplification of TRIM21 and TRIM21 (ΔRING) primers was carried out and the product run on 1% agarose gel (upper panel). Expression of wild-type TRIM21 as well as the TRIM21 (ΔRING) domain was confirmed by Western blotting (lower panel). (B) CHME3 cells were transfected with 4 μg of TRIM21 plasmid or TRIM21 (ΔRING) for 48 h. Cell lysates were resolved on SDS-PAGE and probed with anti-TRIM21, anti-IRF-3 and anti-β- tubulin antibodies by Western blotting. Representative image is shown. (C) Cells transfected with TRIM21 or TRIM21 (ΔRING) were infected with JEV, and total RNA was isolated post 48 h of transfection. Real-time PCR for IFN-β1 was performed, and an average of three independent sets of experiments is plotted and shown. (D) Luciferase assay for IFN-β for cells transfected with TRIM21 or TRIM21 (ΔRING) and infected with JEV along with respective controls was performed. Luciferase activity normalized against β-gal activity was averaged and plotted (*p <0.05, **p < 0.01, ***p < 0.001 from control).

Article Snippet: SiRNA against TRIM21 was purchased from Origene (Rockville, MD, USA) along with Negative control scrambled RNA (TRIM21 Trilencer-27 Human siRNA; #SR304594).

Techniques: Amplification, Agarose Gel Electrophoresis, Expressing, Western Blot, Transfection, Plasmid Preparation, SDS Page, Infection, Isolation, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Control

Journal: Journal of neuroinflammation

Article Title: Regulatory role of TRIM21 in the type-I interferon pathway in Japanese encephalitis virus-infected human microglial cells.

doi: 10.1186/1742-2094-11-24

Figure Lengend Snippet: Figure 4 JEV induces TRIM21 protein levels in human microglial cells in a time-dependent manner. (A) CHME3 cells were either un-infected or infected with JEV at MOI 5 for 6, 12, 24 and 36 h. Cell lysates were resolved on SDS-PAGE and probed with anti-TRIM21 antibody and anti-β- tubulin antibody (loading control) by Western blotting. A representation of three independent experiments is shown along with densitometry analysis with normalization of TRIM21 against β-tubulin, averaging and plotting. (B) CHME3 cells were infected with JEV for 24 h (MOI 5). Total RNA was isolated from harvested cells. cDNA prepared by reverse transcription of control and infected samples was used as a template for qPCR against primers for TRIM21 gene. Average fold change in the TRIM21 mRNA level from three independent experiments is plotted and shown (*p < 0.05, **p < 0.01, ***p < 0.001 from control, #p from 6 h, $p from 12 h).

Article Snippet: SiRNA against TRIM21 was purchased from Origene (Rockville, MD, USA) along with Negative control scrambled RNA (TRIM21 Trilencer-27 Human siRNA; #SR304594).

Techniques: Infection, SDS Page, Control, Western Blot, Isolation, Reverse Transcription

Journal: Journal of neuroinflammation

Article Title: Regulatory role of TRIM21 in the type-I interferon pathway in Japanese encephalitis virus-infected human microglial cells.

doi: 10.1186/1742-2094-11-24

Figure Lengend Snippet: Figure 5 TRIM21 knockdown facilitates JEV-mediated IRF3 activation and upregulation of the IFN-β level. (A) Cells were either transfected with negative control RNA (NC) or transfected with 10nM siRNA against TRIM21 for 48 h. Cell lysates were resolved on SDS-PAGE and probed with anti-TRIM21 antibody and anti-β-tubulin antibody by Western blotting. A representative image is shown. (B) Cells were either non-transfected (C), transfected with negative control RNA (NC) or with TRIM21 siRNA for 24 h followed by JEV infection for 24 h. Cell lysates were resolved on SDS-PAGE and probed with anti-p-IRF3 antibody, anti-IRF3 and anti-β-tubulin antibodies (loading control) by Western blotting. A representative of three independent experiments is shown. Densitometry analyses of Western blot experiments were performed with normalizing p-IRF-3 and p-IRF-3 against β-tubulin. (C) Real-time PCR for IFN-β1 for siRNA-transfected and JEV-infected cells along with the respective controls was performed and averaged for three independent sets of experiments. (D) Luciferase assay for IFN-β for cells transfected with siRNA against TRIM21 and infected with JEV along with respective controls was performed. Luciferase activity normalized against β-gal activity was averaged and plotted (**p < 0.01, ***p < 0.001 from control).

Article Snippet: SiRNA against TRIM21 was purchased from Origene (Rockville, MD, USA) along with Negative control scrambled RNA (TRIM21 Trilencer-27 Human siRNA; #SR304594).

Techniques: Knockdown, Activation Assay, Transfection, Negative Control, SDS Page, Western Blot, Infection, Control, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay

Journal: Journal of neuroinflammation

Article Title: Regulatory role of TRIM21 in the type-I interferon pathway in Japanese encephalitis virus-infected human microglial cells.

doi: 10.1186/1742-2094-11-24

Figure Lengend Snippet: Figure 6 Model showing a plausible role of TRIM21 as a negative regulator of IRF3 activation and IFN-β production following JEV infection in human microglial cells. JEV infection causes activation of the RIG-1 receptor, initiating a downstream signaling mechanism leading to the activation of IRF-3. Phosphorylated IRF-3 dimerizes and translocates into the nucleus, where it leads to the transcription and production of IFN-β. JEV infection also induces the TRIM21 protein, which negatively regulates IRF-3 phosphorylation, leading to reduced IFN-β production. The upregulation of TRIM21 is proposed to be a feedback mechanism to inhibit the innate immune response in JEV infection.

Article Snippet: SiRNA against TRIM21 was purchased from Origene (Rockville, MD, USA) along with Negative control scrambled RNA (TRIM21 Trilencer-27 Human siRNA; #SR304594).

Techniques: Activation Assay, Infection, Phospho-proteomics

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: Science advances

Article Title: CLEC-1 is a death sensor that limits antigen cross-presentation by dendritic cells and represents a target for cancer immunotherapy.

doi: 10.1126/sciadv.abo7621

Figure Lengend Snippet: Fig. 2. Identification of TRIM21 as a ligand of CLEC-1. (A) Representative affinity chromatography elution products (SDS–polyacrylamide gel electrophoresis) after co-IP of UV-treated Raji or Hpb-all whole-cell extract (WCE) with protein G beads coupled to human Fc CLEC-1 or Fc alone (control) (three independent experiments). Arrowheads at 50 kDa indicate a band only present under the Fc CLEC-1 condition. MW, molecular weight. (B) LC-MS analysis (of the 50-kDa gel band) depicting the top hits of Fc CLEC-1 over the Fc alone control condition (n = 4, mean). (C) Representative Western blot of human TRIM21 after co-IP of UV-treated Raji WCE (or without cells as control) with protein G beads coupled to human Fc CLEC-1 or irrelevant Fc CLEC7A (three independent experiments). Arrowheads correspond to TRIM21 detected in the co-IP in the retained and unretained fractions. (D) Representative Western blot of human CLEC-1 in elution product after co-IP of UV-treated Raji WCE and His–CLEC-1 with protein G beads coupled to antihuman TRIM21 mAb and revealed with either anti-His or anti–CLEC-1 mAb (three independent experiments). (E) Representative Western blot of human TRIM21 after co-IP of UV-treated Raji transduced with lentiviral TRIM21 (sh+) or control scrambled shRNA with protein G beads coupled to human Fc CLEC-1 (three independent experiments). (F) Flow cytometry analysis of human Fc CLEC-1 binding on UV-treated Raji transduced with lentiviral TRIM21 or control scrambled shRNA (Ctrl) (data expressed as fold change of binding of Fc CLEC-1 compared to binding of Fc alone) (n = 5, paired t test, *P < 0.05). (G) Sensograms generated by Biacore-based affinity measurement representing interaction of chip-immobilized recombinant human TRIM21 with five increasing concentrations of human Fc CLEC-1 (Fc alone as control) or His–CLEC-1 [irrelevant (irr) protein as control]. (H) Representative ELISA [density of optical (DO)] of recombinant human TRIM21 on coated human Fc CLEC-1 or Fc alone (n = 4, means ± SEM, unpaired t test, **P < 0.01 and ***P < 0.001).

Article Snippet: Reverse co-IP was performed as co-IP, except that beads were coated with anti-TRIM21 antibody (R&D Systems) or control isotype (BD Pharm) and coincubated with His CLEC-1 protein (Bio-Techne).

Techniques: Affinity Chromatography, Polyacrylamide Gel Electrophoresis, Co-Immunoprecipitation Assay, Control, Molecular Weight, Liquid Chromatography with Mass Spectroscopy, Western Blot, Transduction, shRNA, Flow Cytometry, Binding Assay, Generated, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Science advances

Article Title: CLEC-1 is a death sensor that limits antigen cross-presentation by dendritic cells and represents a target for cancer immunotherapy.

doi: 10.1126/sciadv.abo7621

Figure Lengend Snippet: Fig. 6. Treatment with antihuman CLEC-1 antagonist mAb enhances antitumor immunity in CLEC-1 humanized mice. (A) CLEC1A or Clec1a mRNA expression in different subsets of myeloid cells from human or mouse lung tumors from Open-source data of scRNA-seq. AU, arbitrary units. (B) Two-dimensional Uniform Manifold Approximation and Projection and violin plot representations of CLEC1A expression (red scale projection) among clusters of myeloid cells and DCs from scRNA-seq data- set of pan-cancer (breast, colorectal, lung, and ovarian) TME. (C) Box plots representation of log2 [transcript count per million (TMP)+1] values for TRIM21 expression in dif- ferent cancer types from TCGA analyzed by GEPIA tool. BRCA, breast-invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL, cholangiocarcinoma; COAD, colon adenocarcinoma; ESCA, esophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; KIRC, kidney renal clear cell carcinoma; LAML, acute myeloid leukemia; LGG, brain lower-grade glioma; LIHC, liver hepatocellular carcinoma; PAAD, pancreatic adenocarcinoma; READ, rectum adenocarcinoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; TGCT, testicular germ cell tumors. Unpaired t test, *P < 0.05. (D and E) Tumor growth or survival curve of CLEC-1 humanized mice with subcutaneous MC38 adenocarcinoma in combination with CPA (150 mg/kg) (D) or with Hepa 1.6 LIHC (E) and treated with irrelevant isotype control or antihuman CLEC-1 antagonist mAbs #1 or #2 (100 g per mouse) twice a week from days 4 to 21 (n = 14 to 17 of two independent experiments, unpaired t test, *P < 0.05 for tumor growth, and log-rank test, *P < 0.05 and **P < 0.01 for survival). (F) Percentage of PMNs/MDSCs (CD11b+Ly6G+MHC-II−), macrophages (CD11b+F4/80+Ly6C+), DCs (CD11c+MHC-II+), CD69+or Tmem (CD44+) CD8+, or CD4+ (CD3+CD4/8+) T cells evaluated by flow cytometry at day 13 after LIHC in the tumor- burdened livers of CLEC-1 humanized mice treated with isotype control or antihuman CLEC-1 antagonist mAbs #4 (100 g per mouse) (data are expressed in the percentage of CD45+ or in MFI of MHC-II expression) (n = 8 to 13, means ± SEM of three independent experiments, unpaired t test *P < 0.05, **P < 0.01, and ***P < 0.001).

Article Snippet: Reverse co-IP was performed as co-IP, except that beads were coated with anti-TRIM21 antibody (R&D Systems) or control isotype (BD Pharm) and coincubated with His CLEC-1 protein (Bio-Techne).

Techniques: Expressing, Control, Flow Cytometry