Journal: The Journal of Biological Chemistry

Article Title: Opposing roles of E3 ligases TRIM23 and TRIM21 in regulation of ion channel ANO1 protein levels

doi: 10.1016/j.jbc.2021.100738

Figure Lengend Snippet: TRIM21 destabilizes ANO1. A , left , endogenous ANO1 was more markedly decreased after hemagglutinin–TRIM21 expression in TRIM23 KO than WT ZR-75-1 cells. Long and short exposures ( top half ) clearly show TRIM21 effect in TRIM23 WT and KO cells, respectively. GAPDH, loading control. Right , summary data of the left panel. ∗∗ p = 0.0011, n = 5. ANO1 expression was decreased in TRIM23 KO cells. Data are normalized to ANO1 expression without hemagglutinin–TRIM21 expression. B , left , TRIM21 knockdown through RNAi robustly elevated ANO1 expression in ZR-75-1 cells. Right , summary data of the left panel . ∗ p = 0.03 for ANO1, ∗∗ p = 0.005 for TRIM21, n = 4. C , TRIM21 KO increased ANO1 protein but not mRNA level in both the salivary gland and heart relative to the level in sex-matched WT littermates. GFP: reporter of Trim21 transcription in TRIM21 KO mice. Middle , summary data of the left panel ; right , summary data of mRNA levels. ∗ p ≤ 0.033 (n = 7 for the salivary gland, n = 5 for the heart). For mRNA level, p = 0.640, n = 3 for the salivary gland; p = 0.041, n = 3 for the heart. D , TRIM21 KO increased saliva secretion in mice. ∗ p = 0.032, n = 4 (WT/HE: two WT and two heterozygous). E , the heart rate did not differ significantly between TRIM21 KO mice and their WT/HE littermates; p = 0.259, n = 5 (WT/HE: three WT and two heterozygous). ANO1, anoctamin-1; Ctrlsh, control shRNA; RNAi, RNA interference; TRIM, tri partite m otif; TRIM21sh, TRIM21 shRNA.

Article Snippet: The following commercially available antibodies were used: rabbit monoclonal anti-human ANO1 (ab64085) and rabbit polyclonal anti-mouse ANO1 (ab53212), Abcam; rabbit anti-human/mouse TRIM23 (HPA-039605) and mouse anti-FLAG (F1804), Sigma-Aldrich; mouse anti-HA (MMS-101P), Covance Research Products Inc; mouse anti-V5 (P/N-46-0705) and anti-Xpress (R910-25), Invitrogen; and mouse anti-human TRIM21 (sc-25351), anti-ubiquitin (sc-8017), anti-GAPDH (sc-365062), and anti-actin (sc-47778), Santa Cruz Biotechnology.

Techniques: Expressing, Control, Knockdown, shRNA

Journal: The Journal of Biological Chemistry

Article Title: Opposing roles of E3 ligases TRIM23 and TRIM21 in regulation of ion channel ANO1 protein levels

doi: 10.1016/j.jbc.2021.100738

Figure Lengend Snippet: ANO1 physically interacts with TRIM21. A , human ANO1–V5 physically and functionally interacts with human Xpress–TRIM21 in HEK293T cells. Anti-Xpress antibody pulled down ANO1 together with Xpress–TRIM21 from cells coexpressing ANO1–V5 and Xpress-TRIM21 but not from cells expressing ANO1–V5 alone. TRIM21 expression also moderately lowered ANO1 protein expression and increased ANO1 ubiquitination to a limited extent. In IP, the asterisk denotes IgG; the bracket denotes TRIM21 doublet also detected in “Input.” B , exogenous Xpress–TRIM21 in HEK293T cells was captured by GST–ANO1C but not GST alone. Results shown are representative of three independent biological replicates. The asterisk denotes undegraded GST or GST–ANO1. C , endogenous TRIM21 ( red ∗) and ANO1 ( black ∗) interact in ZR-75-1 cells. Upper , endogenous ANO1 was immunoprecipitated with anti-mouse ANO1 or control IgG and then immunoblotted with anti-ANO1 ( top ) and anti-TRIM21 ( bottom ). Lower , endogenous TRIM21 was immunoprecipitated with anti-TRIM21 or control IgG and then immunoblotted with anti-ANO1 ( top ) and anti-TRIM21 ( bottom ). All data in panels A – C are representative of three independent biological replicates. ANO1, anoctamin-1; ANO1–V5, V5-tagged full-length ANO1; ANO1C, ANO1 C terminus; Ig G, immunoglobulin G; TRIM, tri partite m otif; Xpress-TRIM21, Xpress-tagged TRIM21.

Article Snippet: The following commercially available antibodies were used: rabbit monoclonal anti-human ANO1 (ab64085) and rabbit polyclonal anti-mouse ANO1 (ab53212), Abcam; rabbit anti-human/mouse TRIM23 (HPA-039605) and mouse anti-FLAG (F1804), Sigma-Aldrich; mouse anti-HA (MMS-101P), Covance Research Products Inc; mouse anti-V5 (P/N-46-0705) and anti-Xpress (R910-25), Invitrogen; and mouse anti-human TRIM21 (sc-25351), anti-ubiquitin (sc-8017), anti-GAPDH (sc-365062), and anti-actin (sc-47778), Santa Cruz Biotechnology.

Techniques: Expressing, Ubiquitin Proteomics, Immunoprecipitation, Control

Journal: The Journal of Biological Chemistry

Article Title: Opposing roles of E3 ligases TRIM23 and TRIM21 in regulation of ion channel ANO1 protein levels

doi: 10.1016/j.jbc.2021.100738

Figure Lengend Snippet: Schematic showing ANO1 regulation by antagonistic TRIM23 and TRIM21 E3 ligases. Both TRIM23 and TRIM21 bind to ANO1 C terminus. TRIM23 ubiquitinates and stabilizes ANO1 by preventing lysosomal degradation, whereas TRIM21 ubiquitinates and causes ANO1 degradation. EGF signaling enhances ANO1 expression through TRIM23 by a mechanism that remains unclear but potentially involves blocking of TRIM21 ubiquitination of ANO1 (rather than blocking of TRIM21 to the same site—ANO1 C terminus). Green and red dots , antiproteolytic and proteolytic ubiquitination. ANO1, anoctamin-1; EGF, epidermal growth factor; N and C, N- and C-termini; TRIM, tri partite m otif.

Article Snippet: The following commercially available antibodies were used: rabbit monoclonal anti-human ANO1 (ab64085) and rabbit polyclonal anti-mouse ANO1 (ab53212), Abcam; rabbit anti-human/mouse TRIM23 (HPA-039605) and mouse anti-FLAG (F1804), Sigma-Aldrich; mouse anti-HA (MMS-101P), Covance Research Products Inc; mouse anti-V5 (P/N-46-0705) and anti-Xpress (R910-25), Invitrogen; and mouse anti-human TRIM21 (sc-25351), anti-ubiquitin (sc-8017), anti-GAPDH (sc-365062), and anti-actin (sc-47778), Santa Cruz Biotechnology.

Techniques: Expressing, Blocking Assay, Ubiquitin 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: Journal of Virology

Article Title: Human Parvovirus B19 Utilizes Cellular DNA Replication Machinery for Viral DNA Replication

doi: 10.1128/JVI.01881-17

Figure Lengend Snippet: Colocalization of host DNA replication factors with B19V replication centers using immunofluorescence assay and proximity ligation assay (PLA). CD36+ EPCs were infected with B19V. At 24 hpi, cells were cytospun onto slides. (A) Immunofluorescence assay was performed to stain replicating viral genome (BrdU labeled), RPA32, and one of the host DNA replication proteins, Pol δ, Pol α, Pol ε, PCNA, or RFC1, as indicated. STED superresolution microscopy images were taken using a Leica TCS SP8 STED microscope under a 100× objective lens. (B) PLA was performed by costaining the cells with mouse anti-BrdU antibody and one of the rabbit antibodies against Pol δ, Pol α, Pol ε, PCNA, and RFC1, as indicated, which produces amplified signals for interacted proteins in close proximity. Confocal images were taken using an Eclipse C1 Plus confocal microscope (Nikon) that was controlled by Nikon EZ-C1 software. DAPI (4′,6-diamidino-2-phenylindole) was used to stain the nucleus.

Article Snippet: Anti-Pol α (sc-48818) and anti-RFC1 (sc-20993) were purchased from Santa Cruz (Dallas, TX); anti-Pol δ (catalog no. A304-007A-T), anti-total RPA32 (catalog no. A300-244A), anti-RPA32(S4/8) (catalog no. A300-245A-T), and anti-RPA32(S33) (catalog no. A300-246A-T) were from Bethyl Laboratories (Montgomery, TX); anti-Pol ε (catalog no. GTX116557), anti-RPA32(T21) (catalog no. GTX62664), and anti-ATR(pT1989) (catalog no. GTX128145) were from GeneTex (Irvine, CA); anti-PCNA (44434) was from One World Lab (San Diego, CA); and anti-ATM(pS1981) (ab81292) and anti-DNA-PK (ab18192) were from Abcam (Cambridge, MA).

Techniques: Immunofluorescence, Proximity Ligation Assay, Infection, Staining, Labeling, Microscopy, Amplification, Software

Journal: Journal of Virology

Article Title: Human Parvovirus B19 Utilizes Cellular DNA Replication Machinery for Viral DNA Replication

doi: 10.1128/JVI.01881-17

Figure Lengend Snippet: Pol δ and RPA32 play an important role in B19V DNA replication. (A) Western blot analysis of shRNA knockdown. CD36+ EPCs transduced with shRNA-expressing lentiviruses, as indicated, were collected at 2 days postransduction. The cells were analyzed for expression of Pol α, Pol δ, Pol ε, Pol η, Pol λ, and RPA32, as indicated, by Western blotting. β-Actin was used as a loading control. (B) Cell cycle analysis. At 2 days postransduction, shRNA-transduced CD36+ EPCs were labeled with BrdU and analyzed for cell proliferation using flow cytometry with anti-BrdU and DAPI costaining. The percentages of the cells in each phase of the cell cycle are shown as means ± standard deviations (STD) of data obtained from three independent experiments. (C) Southern blot analysis of B19V DNA replication. CD36+ EPCs were transduced with the indicated shRNA for 2 days, and the cells were infected with B19V. At 48 hpi, Hirt DNA was extracted from infected cells and analyzed by Southern blotting with the M20 DNA probe (upper) and the mitochondrial DNA (Mito DNA) probe (lower). (D) B19V progeny virion production. CD36+ EPCs were transduced with the indicated shRNAs for 2 days, and the cells were infected with B19V. After 3 h of incubation, the infected cells were washed twice with phosphate-buffered saline (PBS) and cultured with fresh medium. At 48 hpi, both cells and medium were collected for quantification of B19V virions using qPCR. The value for virus (vgc) produced from shScram-transduced CD36+ EPCs is arbitrarily set at 100. **, P < 0.005; ***, P < 0.001; n.s., not significant.

Article Snippet: Anti-Pol α (sc-48818) and anti-RFC1 (sc-20993) were purchased from Santa Cruz (Dallas, TX); anti-Pol δ (catalog no. A304-007A-T), anti-total RPA32 (catalog no. A300-244A), anti-RPA32(S4/8) (catalog no. A300-245A-T), and anti-RPA32(S33) (catalog no. A300-246A-T) were from Bethyl Laboratories (Montgomery, TX); anti-Pol ε (catalog no. GTX116557), anti-RPA32(T21) (catalog no. GTX62664), and anti-ATR(pT1989) (catalog no. GTX128145) were from GeneTex (Irvine, CA); anti-PCNA (44434) was from One World Lab (San Diego, CA); and anti-ATM(pS1981) (ab81292) and anti-DNA-PK (ab18192) were from Abcam (Cambridge, MA).

Techniques: Western Blot, shRNA, Transduction, Expressing, Cell Cycle Assay, Labeling, Flow Cytometry, Southern Blot, Infection, Incubation, Cell Culture, Produced

Journal: Journal of Virology

Article Title: Human Parvovirus B19 Utilizes Cellular DNA Replication Machinery for Viral DNA Replication

doi: 10.1128/JVI.01881-17

Figure Lengend Snippet: All forms of phosphorylated RPA32 are expressed in B19V-infected cells. CD36+ EPCs were infected with B19V or mock infected. At 48 hpi, the cells were collected and lysed for Western blotting of total RPA32 (A) and of various forms of phosphorylated RPA32 as indicated (B). β-Actin was used as a loading control.

Article Snippet: Anti-Pol α (sc-48818) and anti-RFC1 (sc-20993) were purchased from Santa Cruz (Dallas, TX); anti-Pol δ (catalog no. A304-007A-T), anti-total RPA32 (catalog no. A300-244A), anti-RPA32(S4/8) (catalog no. A300-245A-T), and anti-RPA32(S33) (catalog no. A300-246A-T) were from Bethyl Laboratories (Montgomery, TX); anti-Pol ε (catalog no. GTX116557), anti-RPA32(T21) (catalog no. GTX62664), and anti-ATR(pT1989) (catalog no. GTX128145) were from GeneTex (Irvine, CA); anti-PCNA (44434) was from One World Lab (San Diego, CA); and anti-ATM(pS1981) (ab81292) and anti-DNA-PK (ab18192) were from Abcam (Cambridge, MA).

Techniques: Infection, Western Blot

Journal: Journal of Virology

Article Title: Human Parvovirus B19 Utilizes Cellular DNA Replication Machinery for Viral DNA Replication

doi: 10.1128/JVI.01881-17

Figure Lengend Snippet: All forms of phosphorylated RPA32 are associated with the BrdU-labeled replicating viral genome. CD36+ EPCs were infected with B19V. At 24 hpi, the cells were labeled with BrdU and collected for immunofluorescence (IF) assay with antibodies against RPA32(S4/8), RPA32(T21), RPA32(S33), total RPA32, and anti-BrdU. (A) Images were taken using a Leica TCS SPE confocal microscope at ×63 magnification. (B and C) (B) Images were taken using a Leica TCS SP8 STED superresolution microscope under a 100× objective lens and processed with deconvolution editing using Huygens software. (C) Quantification of the intensity of each stained signaling was performed using Huygens Software.

Article Snippet: Anti-Pol α (sc-48818) and anti-RFC1 (sc-20993) were purchased from Santa Cruz (Dallas, TX); anti-Pol δ (catalog no. A304-007A-T), anti-total RPA32 (catalog no. A300-244A), anti-RPA32(S4/8) (catalog no. A300-245A-T), and anti-RPA32(S33) (catalog no. A300-246A-T) were from Bethyl Laboratories (Montgomery, TX); anti-Pol ε (catalog no. GTX116557), anti-RPA32(T21) (catalog no. GTX62664), and anti-ATR(pT1989) (catalog no. GTX128145) were from GeneTex (Irvine, CA); anti-PCNA (44434) was from One World Lab (San Diego, CA); and anti-ATM(pS1981) (ab81292) and anti-DNA-PK (ab18192) were from Abcam (Cambridge, MA).

Techniques: Labeling, Infection, Immunofluorescence, Microscopy, Software, Staining

Journal: Journal of Virology

Article Title: Human Parvovirus B19 Utilizes Cellular DNA Replication Machinery for Viral DNA Replication

doi: 10.1128/JVI.01881-17

Figure Lengend Snippet: Both phosphorylated and unphosphorylated RPA32 proteins play a role in B19V replication. (A) Expression of RPA32 mutants. Flag-tagged wild-type RPA32 and phosphorylation mutants, namely, RPA32(S4/8A, S4/8D, S33A, S33D, S4/8T21S33 to A or D mutations [all A or D]), were expressed in UT7/Epo-S1 cells by lentivirus transduction. Endogenous RPA32 was knocked down in these cell lines by transducing a shRPA32-expressing lentivirus. Protein expression of RPA32 mutants was detected using anti-RPA32 or anti-Flag antibodies. β-Actin was used as a loading control. Arrowheads indicate endogenous unphosphorylated RPA32. (B) Southern blot analysis of B19V DNA replication. RPA32 mutant-expressing UT7/Epo-S1 cells were cultured under hypoxia conditions and electroporated with SalI-linearized pM20. At 48 hpi, Hirt DNA was extracted from the transfected cells, digested with DpnI enzyme, and analyzed by Southern blotting using the M20 probe (upper panel) and the mitochondrial (Mito) DNA probe (lower panel). (C) B19V virion production. RPA32 mutant-expressing UT7/Epo-S1 cells were cultured under hypoxia conditions for 2 days and electroporated with SalI-linearized M20. At 48 hpi, the cell samples were collected and quantified for B19V virions using qPCR. The value for virus (vgc) produced from M20-transfected RPA32(WT)-expressing UT7/Epo-S1 cells was arbitrarily set at 100. n.s., not significant.

Article Snippet: Anti-Pol α (sc-48818) and anti-RFC1 (sc-20993) were purchased from Santa Cruz (Dallas, TX); anti-Pol δ (catalog no. A304-007A-T), anti-total RPA32 (catalog no. A300-244A), anti-RPA32(S4/8) (catalog no. A300-245A-T), and anti-RPA32(S33) (catalog no. A300-246A-T) were from Bethyl Laboratories (Montgomery, TX); anti-Pol ε (catalog no. GTX116557), anti-RPA32(T21) (catalog no. GTX62664), and anti-ATR(pT1989) (catalog no. GTX128145) were from GeneTex (Irvine, CA); anti-PCNA (44434) was from One World Lab (San Diego, CA); and anti-ATM(pS1981) (ab81292) and anti-DNA-PK (ab18192) were from Abcam (Cambridge, MA).

Techniques: Expressing, Transduction, Southern Blot, Mutagenesis, Cell Culture, Transfection, Produced

Journal:

Article Title: Gram-Positive Bacteria Are Potent Inducers of Monocytic Interleukin-12 (IL-12) while Gram-Negative Bacteria Preferentially Stimulate IL-10 Production

doi:

Figure Lengend Snippet: IL-12 p70 and IL-10 responses by monocytes from nine blood donors stimulated with each of seven gram-positive and seven gram-negative species separately. Concentrations ranged from 5 × 105 to 5 × 107 bacteria/ml. Cytokines were measured in 24-h culture supernatants by ELISA. Each bar represents the mean response of all donors to all gram-positive or gram-negative bacterial strains at a particular concentration. The error bars represent standard errors for the variability between the bacterial species.

Article Snippet: Culture supernatants were diluted 1:5, 1:25, or 1:125 in PBS with 1% BSA and incubated for 2 h together with biotinylated monoclonal mouse IgG1 anti-human IL-12 (clone B-T21; Diaclone [unknown concentration]) diluted 1/100 in PBS with 1% BSA.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal:

Article Title: Gram-Positive Bacteria Are Potent Inducers of Monocytic Interleukin-12 (IL-12) while Gram-Negative Bacteria Preferentially Stimulate IL-10 Production

doi:

Figure Lengend Snippet: IL-12 p70 responses in supernatants after addition of anti-IL-10 antibodies or control IgG1 antibodies to the monocyte cultures before stimulation with 5 × 106 bacteria/ml. IL-12 was measured after 24 h. Bars represent the mean responses of three donors, and the error bars represent standard errors of the means.

Article Snippet: Culture supernatants were diluted 1:5, 1:25, or 1:125 in PBS with 1% BSA and incubated for 2 h together with biotinylated monoclonal mouse IgG1 anti-human IL-12 (clone B-T21; Diaclone [unknown concentration]) diluted 1/100 in PBS with 1% BSA.

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Toxoplasma gondii Type-I ROP18 Targeting Human E3 Ligase TRIM21 for Immune Escape

doi: 10.3389/fcell.2021.685913

Figure Lengend Snippet: Tg ROP18 I interacted with TRIM21’s PRY-SPRY domain. (A) The Co-IP result indicated the interaction of Tg ROP18 I and TRIM21. (B,C) The Co-IP result indicated that ROP18-KD still bound with TRIM21. (D) Sketch map of TRIM21 WT and truncation mutants. (E) The Co-IP results showed the PRY-SPRY domain of TRIM21 was indispensable for Tg ROP18 I -TRIM21 interaction. The experiments were repeated three times. The values were analyzed using the one-way ANOVA. Data were expressed as the mean ± SEM (** p < 0.01; *** p < 0.001; IP, immunoprecipitation).

Article Snippet: HEK293T cells were seeded in T25 flasks to 90% confluence, and then transfected with pcDNA3.1-TRIM21-HA for 24 h, or treated with 100 U/ml IFN-γ for 0, 12, or 24 h. The cells were then harvested and the cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) or anti-p65 mouse monoclonal antibody (Cell Signaling Technology, MA, United States).

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Toxoplasma gondii Type-I ROP18 Targeting Human E3 Ligase TRIM21 for Immune Escape

doi: 10.3389/fcell.2021.685913

Figure Lengend Snippet: Tg ROP18 I promoted the phosphorylation of TRIM21. (A,B) HEK293T cells were transfected with different amount of pcDNA3.1-ROP18 I -FLAG as indicated, and the cell lysates were subjected to TRIM21 IP and Western blotting. The more plasmids were transfected to HEK293T cells, the higher levels of phosphorylated TRIM21 were observed, and the differences between groups were significant. (C,D) HFF cells were infected with CEP or CEP- rop18 I , and then the total protein was extracted and subjected to TRIM21 IP and Western blotting. The results showed that more phosphorylated TRIM21 was detected in the CEP- rop18 I infected cells than which detected in the CEP infected cells and uninfected cells, while the phosphorylation levels of TRIM21 were not significantly different between the CEP infected and uninfected cells. The experiments were repeated three times. The values were analyzed using the one-way ANOVA. Data were expressed as the mean ± SEM (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: HEK293T cells were seeded in T25 flasks to 90% confluence, and then transfected with pcDNA3.1-TRIM21-HA for 24 h, or treated with 100 U/ml IFN-γ for 0, 12, or 24 h. The cells were then harvested and the cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) or anti-p65 mouse monoclonal antibody (Cell Signaling Technology, MA, United States).

Techniques: Phospho-proteomics, Transfection, Western Blot, Infection

Journal: Frontiers in Cell and Developmental Biology

Article Title: Toxoplasma gondii Type-I ROP18 Targeting Human E3 Ligase TRIM21 for Immune Escape

doi: 10.3389/fcell.2021.685913

Figure Lengend Snippet: Tg ROP18 I promoted TRIM21 degradation through lysosomal pathway. (A) HEK293T cells were transfected with the increased amounts of pcDNA3.1-ROP18 I -FLAG as indicated. (B) HEK293T cells were co-transfected with a stable amount of pcDNA3.1-TRIM21-HA and the increased amounts of pcDNA3.1-ROP18 I -FLAG as indicated. The endogenous or overexpressed TRIM21 level was decreased with the increased ROP18 level. (C) HEK293T cells were co-transfected with pcDNA3.1-TRIM21-HA and pcDNA3.1-ROP18 I -FLAG or pcDNA3.1-ROP18 I -KD-FLAG as indicated. The results of Western blotting detection with the cell lysates indicated that much more TRIM21 was detected in the ROP18-KD overexpression group than in the ROP18 overexpression group. (D) Lysates of HFFs infected with RH or RH-△ rop18 was detected by Western blotting, and more TRIM21 was detected in the RH-△ rop18 infection group than in the RH infection group. (E) HEK293T cells were co-transfected with 1mg of pcDNA3.1-TRIM21-HA and increased amounts of pcDNA3.1-ROP18 I -FLAG. The cells were treated with MG132 or Leupeptin, or left untreated. Cell lysates were subjected to Western blotting, and the results showed that TRIM21’s level was decreased with the increased amount of Tg ROP18 I in the cells treated with MG132 or DMSO. However, TRIM21’s level was kept stable in the Leupeptin treated group. All the experiments were repeated three times. IB, immunoblot.

Article Snippet: HEK293T cells were seeded in T25 flasks to 90% confluence, and then transfected with pcDNA3.1-TRIM21-HA for 24 h, or treated with 100 U/ml IFN-γ for 0, 12, or 24 h. The cells were then harvested and the cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) or anti-p65 mouse monoclonal antibody (Cell Signaling Technology, MA, United States).

Techniques: Transfection, Western Blot, Over Expression, Infection

Journal: Frontiers in Cell and Developmental Biology

Article Title: Toxoplasma gondii Type-I ROP18 Targeting Human E3 Ligase TRIM21 for Immune Escape

doi: 10.3389/fcell.2021.685913

Figure Lengend Snippet: TRIM21 transcription and translation levels were upregulated following T. gondii infection. HFF cells infected with RH or CEP were harvested for detection of TRIM21 transcription and translation level. (A) Comparison of the TRIM21 transcription levels between the indicated groups with qRT-PCR. The CEP infection resulted in the highest TRIM21 transcription level, followed by RH infection, and uninfection at 1 h post infection, the differences between groups were significant. At 24 h post infection, the RH infection resulted in a significant higher TRIM21 transcription level than in CEP infection or uninfection groups between which no significant difference was observed in TRIM21 transcription level. (B,C) Comparison of the translation level of TRIM21 in the HFF cells infected with RH or CEP for the indicated time with Western blot (up panels). The densitometrical analysis for the intensity of TRIM21 bands normalized to its corresponding GAPDH intensity showed that, both CEP and RH infection resulted in significant higher transcription levels of TRIM21 than in uninfected cells (down panels). All the experiments were repeated four times. The values were analyzed using the one-way ANOVA. Data were expressed as the mean ± SEM (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: HEK293T cells were seeded in T25 flasks to 90% confluence, and then transfected with pcDNA3.1-TRIM21-HA for 24 h, or treated with 100 U/ml IFN-γ for 0, 12, or 24 h. The cells were then harvested and the cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) or anti-p65 mouse monoclonal antibody (Cell Signaling Technology, MA, United States).

Techniques: Infection, Comparison, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: Toxoplasma gondii Type-I ROP18 Targeting Human E3 Ligase TRIM21 for Immune Escape

doi: 10.3389/fcell.2021.685913

Figure Lengend Snippet: TRIM21 is involved in the IFN-γ induced inhibition of CEP proliferation (but not RH proliferation) in HFFs. The proliferation of RH and CEP tachyzoies in the HFFs was evaluated after stimulation with IFN-γ for 24 h (A–C) , or transfection with pcDNA3.1-TRIM21-HA for 24 h (E–F) ; and proliferation of CEP-WT was evaluated after transfection with si-TRIM21 for 48 h in HFFs followed by IFN-γ treatment for 24 h (H–I) . TRIM21 protein levels were measured by Western blotting (A,D,G) . The average number of tachyzoites in 100 parasitophorous vacuoles (PVs) was counted (B,E,H) , and the percentage of the PVs containing 1, 2, 4, or 8 parasites was determined by immune fluorescence assay (C,F,I) . (A) The TRIM21 protein level was significantly up-regulated under IFN-γ stimulation in a dose-dependent manner, with the response concentration ranged from 50 to 500 U/ml. (B,C) The proliferation of the RH and CEP tachyzoites in HFFs was significantly inhibited after IFN-γ stimulation. (D) The overexpression of TRIM21 was detected. (E,F) The overexpression of TRIM21 significantly inhibited the CEP proliferation, but not affected RH proliferation. (G) The si-TRIM21 transfection significantly inhibited the TRIM21 translation. (H,I) The IFN-γ induced inhibition of CEP proliferation was relieved by TRIM21 knockdown. All the experiments were repeated three times. The values were analyzed using the one-way ANOVA and two-way ANOVA. Data were expressed as the mean ± SEM (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: HEK293T cells were seeded in T25 flasks to 90% confluence, and then transfected with pcDNA3.1-TRIM21-HA for 24 h, or treated with 100 U/ml IFN-γ for 0, 12, or 24 h. The cells were then harvested and the cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) or anti-p65 mouse monoclonal antibody (Cell Signaling Technology, MA, United States).

Techniques: Inhibition, Transfection, Western Blot, Fluorescence, Concentration Assay, Over Expression, Knockdown

Journal: Frontiers in Cell and Developmental Biology

Article Title: Toxoplasma gondii Type-I ROP18 Targeting Human E3 Ligase TRIM21 for Immune Escape

doi: 10.3389/fcell.2021.685913

Figure Lengend Snippet: TRIM21 knockdown relieves the IFN-γ-induced ubiquitin labeling on CEP parasitophorous vacuole membrane (PVM) in HFFs. (A,B) HFFs were transfected with negative control siRNA (si-NC) or siRNA specific against TRIM21 (si-TRIM21), and stimulated with IFN-γ or not as indicated. After T. gondii infection, HFFs were subjected to immunofluorescence assay (IFA). IFN-γ induced ubiquitin labeling on the CEP PVM, but this labeling was relieved by TRIM21 knockdown regardless of IFN-γ simulation or not. (C,D) HFFs were stimulated with IFN-γ or not, and infected with CEP. The cells were then treated with LysoTracker ® (acidic dye) and subjected to immunofluorescence assay (IFA). The result showed us that IFN-γ induced acidification of CEP. On the left, a representative fluorescent image is shown for the T. gondii CEP strain expressing GFP. The yellow box inside each representative image is shown as magnified pictures nearby (A,C) . The percentage of vacuoles stained red with ubiquitin labeling or LysoTracker ® was shown in the right bar diagram (B,D) . Scale bar is 10 μm. The experiments were repeated three times. The values were analyzed using the one-way ANOVA or two-tailed unpaired Student t test. Data were expressed as the mean ± SEM (** p < 0.01; *** p < 0.001).

Article Snippet: HEK293T cells were seeded in T25 flasks to 90% confluence, and then transfected with pcDNA3.1-TRIM21-HA for 24 h, or treated with 100 U/ml IFN-γ for 0, 12, or 24 h. The cells were then harvested and the cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) or anti-p65 mouse monoclonal antibody (Cell Signaling Technology, MA, United States).

Techniques: Knockdown, Ubiquitin Proteomics, Labeling, Membrane, Transfection, Negative Control, Infection, Immunofluorescence, Expressing, Staining, Two Tailed Test

Journal: Frontiers in Cell and Developmental Biology

Article Title: Toxoplasma gondii Type-I ROP18 Targeting Human E3 Ligase TRIM21 for Immune Escape

doi: 10.3389/fcell.2021.685913

Figure Lengend Snippet: Tg ROP18 I relieved TRIM21 mediated inhibition of T. gondii proliferation regardless of strain types. (A–D) HFFs were transfected with pcDNA3.1-TRIM21-HA or pcDNA3.1(+) for control, and infected with RH-△ rop18 (A,B) or CEP- rop18 I (C,D) parasites as indicated. Parasitic proliferation was measured at 18 h (RH-△ rop18 ) or 24 h (CEP- rop18 I ) post-infection. The average number of tachyzoites in 100 vacuoles (A,C) or the number of vacuoles containing 1, 2, 4, or 8 parasites (B,D) was determined by fluorescence microscopy. The results indicated that TRIM21 overexpression inhibited the RH-△ rop18 multiplication, but had no significant effect on CEP- rop18 I multiplication. The experiments were repeated three times. The values were analyzed using the two-tailed unpaired Student t test and two-way ANOVA. Data were expressed as the mean ± SEM (** p < 0.01).

Article Snippet: HEK293T cells were seeded in T25 flasks to 90% confluence, and then transfected with pcDNA3.1-TRIM21-HA for 24 h, or treated with 100 U/ml IFN-γ for 0, 12, or 24 h. The cells were then harvested and the cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) or anti-p65 mouse monoclonal antibody (Cell Signaling Technology, MA, United States).

Techniques: Inhibition, Transfection, Control, Infection, Fluorescence, Microscopy, Over Expression, Two Tailed Test

Journal: Frontiers in Cell and Developmental Biology

Article Title: Toxoplasma gondii Type-I ROP18 Targeting Human E3 Ligase TRIM21 for Immune Escape

doi: 10.3389/fcell.2021.685913

Figure Lengend Snippet: TRIM21 mediated NF-κB activation. (A,B) In the HFF cells stimulated with the indicated concentrations of IFN-γ for 24 h, the p-p65 (S536) level was significantly higher than that in the untreated group. (C,D) Western-blot detection of p-p65 (S536) level showed that TRIM21 overexpression significantly elevated p-p65 (S536) phosphorylation in dose-dependent manner. (E,F) In the siRNA-TRIM21 transfected groups, TRIM21 expression was suppressed, but no significant difference was found in the p-p65 (S536) levels between the TRIM21 knockdown group with or without IFN-γ induction. The experiments were repeated three times. The values were analyzed using the one-way ANOVA and the data were expressed as the mean ± SEM (** p < 0.01; *** p < 0.001).

Article Snippet: HEK293T cells were seeded in T25 flasks to 90% confluence, and then transfected with pcDNA3.1-TRIM21-HA for 24 h, or treated with 100 U/ml IFN-γ for 0, 12, or 24 h. The cells were then harvested and the cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) or anti-p65 mouse monoclonal antibody (Cell Signaling Technology, MA, United States).

Techniques: Activation Assay, Western Blot, Over Expression, Phospho-proteomics, Transfection, Expressing, Knockdown

Journal: Frontiers in Cell and Developmental Biology

Article Title: Toxoplasma gondii Type-I ROP18 Targeting Human E3 Ligase TRIM21 for Immune Escape

doi: 10.3389/fcell.2021.685913

Figure Lengend Snippet: The interaction of p65 and IκB-α was suppressed with TRIM21 overexpression. Interaction of TRIM21 and IκB-α in the total cell lysates of HEK293T cells were analyzed by IP and Western-blot after either treatment with IFN-γ or transfection with pcDNA3.1-TRIM21-HA for the indicated time. (A–C) The IP with the anti-TRIM21 antibody identified the interaction of IκB-α with the overexpressed and the endogenous TRIM21. TRIM21 overexpression promoted TRIM21-IκB-α interaction (A) . IFN-γ induction elevated TRIM21 production and promoted TRIM21-IκB-α interaction (B) . The TRIM21 production induced by IFN-γ increased with the prolonged treating time and promoted TRIM21-IκB-α interaction (C) . (D,E) The IP with the anti-p65 antibody identified the complex of IκB-α-TRIM21-p65 (D) , and the interaction of p65-ubiquitin (E) . The experiments were repeated three times. The TRIM21 band is indicated with “*”, and the IκB-α band is indicated with a black arrow.

Article Snippet: HEK293T cells were seeded in T25 flasks to 90% confluence, and then transfected with pcDNA3.1-TRIM21-HA for 24 h, or treated with 100 U/ml IFN-γ for 0, 12, or 24 h. The cells were then harvested and the cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) or anti-p65 mouse monoclonal antibody (Cell Signaling Technology, MA, United States).

Techniques: Over Expression, Western Blot, Transfection, Ubiquitin Proteomics

Journal: Frontiers in Cell and Developmental Biology

Article Title: Toxoplasma gondii Type-I ROP18 Targeting Human E3 Ligase TRIM21 for Immune Escape

doi: 10.3389/fcell.2021.685913

Figure Lengend Snippet: Tg ROP18 I targets host TRIM21 for immune escape. 1. Host IFN-γ-induced factor TRIM21 restricted T. gondii replication through NF-κB activation and TRIM21 overexpression suppressed the p65-IκB-α interaction to activate NF-κB pathway. 2. Tg ROP18 I which was discharged by T. gondii , interacted with the PRY-SPRY domain of human TRIM21, promoted TRIM21 phosphorylation, and induced TRIM21 degradation via lysosomal pathway. 3. IFN-γ induced ubiquitin labeling on the CEP PVM which resulted in PV acidification and death of parasites, but this labeling was relieved by TRIM21 knockdown regardless of IFN-γ simulation or not.

Article Snippet: HEK293T cells were seeded in T25 flasks to 90% confluence, and then transfected with pcDNA3.1-TRIM21-HA for 24 h, or treated with 100 U/ml IFN-γ for 0, 12, or 24 h. The cells were then harvested and the cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) or anti-p65 mouse monoclonal antibody (Cell Signaling Technology, MA, United States).

Techniques: Activation Assay, Over Expression, Phospho-proteomics, Ubiquitin Proteomics, Labeling, Knockdown

Journal: bioRxiv

Article Title: Hybrid female sterility due to cohesin protection errors in oocytes

doi: 10.1101/2025.02.16.638358

Figure Lengend Snippet: ( A ) Phylogenetic tree of mouse species (myr, million years) and schematic of the hybrid mouse system to study hybrid incompatibility in female meiosis. ( B ) 6-month fertility test from indicated genotypes. Age-matched domesticus (dom), spicilegus (spi) and the hybrid female (F1 hybrid) were used. Each column represents the number of pups per litter for each breeding cage (n = 4 breeding cages per genotype). Note that two F1 female hybrids gave birth to one pup each, which died within few days after the birth. ( C ) Schematic of meiotic chromosome segregation. Cohesin is initially loaded along the chromosome axes and holds sister chromatids together. Cohesin cleavage along the chromosome arm at anaphase I allows the segregation of homologous chromosomes. During metaphase II, sister chromatids maintain their cohesion by the residual cohesin at the pericentromere. Cleavage of this remaining cohesin at anaphase II leads to sister chromatid segregation. ( D ) DNA was visualized in domesticus and hybrid oocytes by incubating with SPY-DNA or expressing the Separase biosensor, H2B-mScarlet-Rad21- mNeonGreen (see ) to live-image anaphase I; the mScarlet images are shown in the figure; PB, polar body; dashed lines, oocyte cortex. ( E ) Anaphase chromosome lagging rates in D were quantified (n = 68 and 65 oocytes for domesticus and hybrid, respectively); red lines, mean; unpaired two-tailed t test was used for statistical analysis; ** P <0.01. ( F ) Chromosome spreads were performed at metaphase II using domesticus , spicilegus and hybrid oocytes and stained for HEC1 and REC8 (right); orange arrowhead, univalents; white arrowhead, bivalents. ( G ) The number of bivalents per egg and the percentage of eggs with cohesin along the chromosome axes were quantified using the images in F (left bottom, n = 13, 13, and 13 eggs for domesticus , spicilegus , and hybrid); each dot in the graph represents a single egg; red line, median. ( H ) domesticus and hybrid oocytes expressing mCherry-Trim21 with or without the anti-REC8 antibody were fixed at metaphase II and stained for ACA (centromere). The percentage of meiosis II eggs with >1 bivalent, >1 precocious separated sister chromatids (PSSC), and normal univalents were quantified (n = 12, 10, 16, and 18 eggs for domesticus + TRIM21, domesticus + TRIM21 + anti-REC8, hybrid + TRIM21, and hybrid + TRIM21+anti- REC8); scale bars, 5 µ m. Schematics in A and C were created using BioRender.

Article Snippet: GV-intact prophase I oocytes were microinjected with ∼5 pl of cRNA or antibodies in M2 containing 5 µM milrinone, using a micromanipulator TransferMan 4r and FemtoJet 4i (Eppendorf). cRNA used for microinjections were Egfp-Bub1 ( M. m. domesticus or P. maniculatus bairdii BUB1 fused with EGFP at the N-terminus, 1184 and 950 ng/µl, respectively), mCherry-Trim21 (Addgene cat# 105522, M. musculus domesticus TRIM21 fused with mCherry at the C-terminus, 1500 or 3000 ng/µl for BUB1 and REC8 TrimAway, respectively), and hH2B-mScarlet-hRad21-mNeonGreen (Separase sensor, pNM853, human H2B fused with human Rad21(142-476 a.a.) at the C-terminus with the Rad21 fragment flanked by two fluorescent proteins, mScarlet and mNeonGreen, 750 ng/µl).

Techniques: Expressing, Two Tailed Test, Staining

Journal: bioRxiv

Article Title: Hybrid female sterility due to cohesin protection errors in oocytes

doi: 10.1101/2025.02.16.638358

Figure Lengend Snippet: ( A ) Chromosome spreads were performed at metaphase I using domesticus , spicilegus , and hybrid oocytes and stained for BUB1. Line scans were performed to quantify BUB1 signal intensities along the chromosome arm starting from the centromere. Line graph shows the mean values of BUB1 intensity along the chromosome arm with the shades representing standard deviation (n = 26, 36, and 60 chromosomes for domesticus , spicilegus , and hybrid). The enlarged area of the graph highlights the difference of BUB1 intensity along the chromosome arm. ( B ) Chromosome spreads were performed at metaphase I using domesticus , spicilegus , and hybrid oocytes and stained for H2ApT121. Graph is the quantification of chromosomal H2ApT121 signal intensities per oocyte. (n = 13, 6, and 17 oocytes for domesticus , spicilegus , and hybrid); each dot in the graph represents a single oocyte; red line, median. ( C ) Ovarian granulosa cells from domesticus , spicilegus , and hybrid were fixed and stained for BUB1 and H2ApT121. Line graph is the quantification of BUB1 and H2ApT121 signal intensities along the chromosome arm starting from the centromere. Lines indicate the mean values of BUB1 and H2ApT121 intensities, and the shades represents standard deviation (n = 64, 93, and 102 chromosomes for domesticus , spicilegus , and hybrid). ( D ) domesticus and hybrid oocytes expressing mCherry-TRIM21 with the control IgG or the anti-BUB1 antibodies were fixed at metaphase II and stained for ACA. The percentage of eggs with >1 bivalent, >1 precocious separated sister chromatids (PSSC), and normal univalents were quantified (n = 21, 26, 38, and 54 eggs for domesticus + TRIM21 + IgG, domesticus + TRIM21 + anti-BUB1, hybrid + TRIM21 + IgG, and hybrid + TRIM21 + anti- BUB1). ( E ) domesticus oocytes expressing EGFP-BUB1 derived from domesticus or Peromyscus maniculatus were fixed at metaphase II and stained for EGFP. The percentage of meiosis II eggs with >1 bivalent was quantified (n = 31, 26, 39 oocytes for control, domesticus EGFP-BUB1, Peromyscus EGFP-BUB1, respectively); each dot in the graph represents a single oocyte; red line, mean; unpaired two-tailed t test was used for statistical analysis; ** P <0.01, ** *P <0.001; scale bars, 5 µ m.

Article Snippet: GV-intact prophase I oocytes were microinjected with ∼5 pl of cRNA or antibodies in M2 containing 5 µM milrinone, using a micromanipulator TransferMan 4r and FemtoJet 4i (Eppendorf). cRNA used for microinjections were Egfp-Bub1 ( M. m. domesticus or P. maniculatus bairdii BUB1 fused with EGFP at the N-terminus, 1184 and 950 ng/µl, respectively), mCherry-Trim21 (Addgene cat# 105522, M. musculus domesticus TRIM21 fused with mCherry at the C-terminus, 1500 or 3000 ng/µl for BUB1 and REC8 TrimAway, respectively), and hH2B-mScarlet-hRad21-mNeonGreen (Separase sensor, pNM853, human H2B fused with human Rad21(142-476 a.a.) at the C-terminus with the Rad21 fragment flanked by two fluorescent proteins, mScarlet and mNeonGreen, 750 ng/µl).

Techniques: Staining, Standard Deviation, Expressing, Control, Derivative Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Hybrid female sterility due to cohesin protection errors in oocytes

doi: 10.1101/2025.02.16.638358

Figure Lengend Snippet: ( A ) Phylogenetic tree of the Mus , Rattus , and Peromyscus genera (myr, million years) and schematic of the Peromyscus hybrid mouse system. ( B ) P. maniculatus , P. polionotus , and their hybrid oocytes (with or without the EGFP-BUB1 expression) were matured to meiosis II, fixed and stained for HEC1. The numbers in the DNA images indicate the number of split sister chromatids in the egg. ( C ) Graph shows the quantification of the number of separated sister chromatids in each meiosis II egg (n = 162, 171, 147, and 50 eggs for P. maniculatus , P. polionotus , hybrid, and hybrid + EGFP-BUB1, respectively); each dot represents an independent experiment; bars and error bars represent mean and standard deviation, respectively. ( D ) P. maniculatus , P. polionotus , and hybrid metaphase II eggs were fixed and stained for REC8 and HEC1. ( E ) Bar graph shows the proportion of meiosis II eggs with centromeric REC8 signals in each genotype (n = 32, 21, and 40 eggs for P. maniculatus , P. polionotus , and the hybrid); each dot represents an independent experiment; bars and error bars represent mean and standard deviation, respectively; unpaired two-tailed t test was used for statistical analysis. Images from were used for the quantification. Dot plot is a quantification of sister-kinetochore distance at metaphase II; images from were used for the quantification; red line, median; Mann-Whitney test was used for statistical analysis. ( F-H ) P. maniculatus , P. polionotus , and hybrid oocytes were fixed at metaphase I and stained for ACA together with PP2A (f), H2ApT121 (g), or BUB1 (h). The graphs show the quantification of centromeric signal intensities for PP2A (f, n = 678, 403, and 448 centromeres for P. maniculatus , P. polionotus , and the hybrid), H2ApT121 (g, n = 473, 301, and 322 centromeres for P. maniculatus , P. polionotus , and the hybrid), and BUB1 (h, n = 805, 394, and 1213 centromeres for P. maniculatus , P. polionotus , and the hybrid); each dot represents a single centromere; red line, median; Mann-Whitney test was used for statistical analysis. **P <0.01, ****P <0.0001; scale bars, 5 µ m.

Article Snippet: GV-intact prophase I oocytes were microinjected with ∼5 pl of cRNA or antibodies in M2 containing 5 µM milrinone, using a micromanipulator TransferMan 4r and FemtoJet 4i (Eppendorf). cRNA used for microinjections were Egfp-Bub1 ( M. m. domesticus or P. maniculatus bairdii BUB1 fused with EGFP at the N-terminus, 1184 and 950 ng/µl, respectively), mCherry-Trim21 (Addgene cat# 105522, M. musculus domesticus TRIM21 fused with mCherry at the C-terminus, 1500 or 3000 ng/µl for BUB1 and REC8 TrimAway, respectively), and hH2B-mScarlet-hRad21-mNeonGreen (Separase sensor, pNM853, human H2B fused with human Rad21(142-476 a.a.) at the C-terminus with the Rad21 fragment flanked by two fluorescent proteins, mScarlet and mNeonGreen, 750 ng/µl).

Techniques: Expressing, Staining, Standard Deviation, Two Tailed Test, MANN-WHITNEY

Journal: International journal of biological sciences

Article Title: Deacetylation of Glutaminase by HDAC4 contributes to Lung Cancer Tumorigenesis.

doi: 10.7150/ijbs.69882

Figure Lengend Snippet: Figure 4. TRIM21 is the E3 ligase for GAC. A, B Indicated plasmids were transfected into H1299 cells. Interaction between GAC and TRIM21 were detected by immunoprecipitation and western blot assay. C-E Indicated plasmids were transfected into H1299 cells. The levels of GAC ubiquitination were detected by immunoprecipitation and western blot assay. F-H Indicated plasmids and siRNAs were transfected into H1299 cells. The levels of GAC ubiquitination were detected by immunoprecipitation and western blot assay. WCL: whole cell lysate. I Indicated siRNAs were transfected into H1299 cells. The protein expression was determined by western blot and glutaminase activity assay was performed. J Indicated plasmids were transfected into H1299 cells. The protein expression were determined by western blot and glutaminase activity assay was performed. Data are showed as mean ± SD, n=3. **P < 0.01.

Article Snippet: The HDAC4 siRNAs (OriGene, SR306523) and TRIM21 siRNAs (OriGene, SR304594) were purchased from OriGene.

Techniques: Transfection, Immunoprecipitation, Western Blot, Ubiquitin Proteomics, Expressing, Activity Assay

Journal: International journal of biological sciences

Article Title: Deacetylation of Glutaminase by HDAC4 contributes to Lung Cancer Tumorigenesis.

doi: 10.7150/ijbs.69882

Figure Lengend Snippet: Figure 5. Lys311 acetylation promotes GAC-TRIM21 interaction and GAC ubiquitination. A Indicated plasmids were transfected into H1299 cells. Interaction between GAC and TRIM21 were detected by immunoprecipitation and western blot assay. B Indicated plasmids were transfected into H1299 cells followed by treatment with or without NAM and TSA. Interaction between GAC and TRIM21 were detected by immunoprecipitation and western blot assay. C Indicated plasmids were transfected into H1299 cells. Interaction between GAC and TRIM21 were detected by immunoprecipitation and western blot assay. D Indicated plasmids were transfected into H1299 cells. Interaction between GAC and TRIM21 were detected by immunoprecipitation and western blot assay. E Indicated plasmids were transfected into H1299 cells. The levels of GAC ubiquitination were detected by immunoprecipitation and western blot assay. WCL: whole cell lysate.

Article Snippet: The HDAC4 siRNAs (OriGene, SR306523) and TRIM21 siRNAs (OriGene, SR304594) were purchased from OriGene.

Techniques: Ubiquitin Proteomics, Transfection, Immunoprecipitation, Western Blot

Journal: International journal of biological sciences

Article Title: Deacetylation of Glutaminase by HDAC4 contributes to Lung Cancer Tumorigenesis.

doi: 10.7150/ijbs.69882

Figure Lengend Snippet: Figure 8. A working model of GAC acetylation in NSCLC. A working model depicting the molecular mechanism of HDAC4 mediated GAC deacetylation and TRIM21 mediated GAC ubiquitination to regulation tumorigenesis in NSCLC.

Article Snippet: The HDAC4 siRNAs (OriGene, SR306523) and TRIM21 siRNAs (OriGene, SR304594) were purchased from OriGene.

Techniques: Ubiquitin 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 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: The Journal of International Medical Research

Article Title: Sjögren’s syndrome positive for isolated anti-Ro52/SS-A antibody and anti-centromere antibody

doi: 10.1177/03000605241293986

Figure Lengend Snippet: Clinical characteristics of five cases of isolated anti-Ro52/SS-A antibody-positive Sjögren’s syndrome.

Article Snippet: The result of an enzyme-linked immunosorbent assay (ELISA) for Ro52/SS-A antigen (Orgentec Diagnostika, Mainz, Germany) in the patient’s serum was positive at 28.3 U/ml (the control dilution medium concentration was 0.4 U/ml).

Techniques: Isolation, Enzyme-linked Immunosorbent Assay