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: PLOS Pathogens

Article Title: TRIM21 restricts influenza A virus replication by ubiquitination-dependent degradation of M1

doi: 10.1371/journal.ppat.1011472

Figure Lengend Snippet: (A) Mass spectrometry identification of TRIM21 as a potential binding partner of H9N2 M1. A549 cells infected with H9N2 virus at an MOI = 1.0 for 24 h were immunoprecipitated with anti-M1 mAb or mouse IgG, and analyzed by mass spectrometry. The TRIM21 was detected in anti-M1 IP samples. Red indicates matched B ions, blue indicates matched Y ions, green indicates unmatched ions, grey indicates precursor ions. (B) TRIM21 interacts with H9N2 M1. Myc-tagged TRIM21, Flag-GST-tagged H9N2 M1 plasmids were transfected into HEK293T cells individually. Cell lysates were subjected to coimmunoprecipitation and western blotting using the indicated antibodies. (C) Endogenous association of TRIM21 with H9N2 M1. A549 cells infected with H9N2 virus at an MOI = 1.0 for 24 h were immunoprecipitated with anti-M1 or control IgG, and analyzed by western blotting with the indicated antibodies. (D) TRIM21 interacts directly with H9N2 M1. Purified His-H9-M1 protein mixed with the GST or GST-TRIM21 proteins was pulled down with GST Sepharose, and analyzed by western blotting with the corresponding antibodies. (E) TRIM21 colocalizes with H9N2 M1. HEK293T cells were transfected with Flag-tagged TRIM21 plasmid for 24 h, infected with H9N2 virus at an MOI = 1.0 for 12 h, or co-tranfected with Flag-tagged TRIM21 and Myc-tagged H9 M1 for 24h, and then incubated with the anti-Flag rabbit mAb, anti-Myc mouse mAb, anti-M1 mouse mAb, FITC-Labeled goat anti-rabbit IgG, and Alexa Fluor 546-conjugated donkey anti-mouse IgG. DAPI staining revealed the nuclei. The images were obtained by confocal microscopy. Scale bar = 10 mm. (F) TRIM21 interacts with H3N2 M1 and H5N1 M1. Myc-tagged TRIM21 and Flag-GST-tagged PR8 M1 or H3N2 M1, H5N1 M1, H7N9 M1 plasmids were individually transfected into HEK293T cells. Cell lysates were subjected to coimmunoprecipitation and western blotting using the indicated antibodies. (G) Endogenous association of TRIM21 with H3N2 M1 and H5N1 M1. A549 cells infected with PR8, H3N2, H5N1 and H7N9 viruses at an MOI = 1.0 for 24 h were immunoprecipitated with anti-M1 or control IgG, and analyzed by western blotting with the indicated antibodies. Each experiment was independently performed with three biological repeats.

Article Snippet: Rabbit mAbs against TRIM21 (12108-1-AP) was purchased from Proteintech Group, Inc. (Manchester, UK).

Techniques: Mass Spectrometry, Binding Assay, Infection, Virus, Immunoprecipitation, Transfection, Western Blot, Control, Purification, Plasmid Preparation, Incubation, Labeling, Staining, Confocal Microscopy

Journal: PLOS Pathogens

Article Title: TRIM21 restricts influenza A virus replication by ubiquitination-dependent degradation of M1

doi: 10.1371/journal.ppat.1011472

Figure Lengend Snippet: (A) Amino acid sequence alignment of the M1 protein from various IAV, including PR8, H3N2, H5N1, H7N9, H9N2, H5Nx, and H7Nx. (B) The structural prediction of the M1 proteins from PR8, H3N2, H5N1, H7N9, and H9N2 viruses using SWISS MODEL and PyMOL software. (C-E) R 95 of M1 is required for the TRIM21 interaction. HEK293T cells were separately transfected with plasmids encoding Flag-GST H3 M1, H5 M1, H9 M1 and its mutants (T 37 A, R 95 K, S 242 N, K 242 N), Myc-tagged TRIM21 plasmid for 48 h individually, and then cell lysates were incubated as indicated for a coimmunoprecipitation assay and analyzed by western blotting with the indicated antibodies. Each experiment was independently performed with three biological repeats.

Article Snippet: Rabbit mAbs against TRIM21 (12108-1-AP) was purchased from Proteintech Group, Inc. (Manchester, UK).

Techniques: Sequencing, Structural Proteomics, Software, Transfection, Plasmid Preparation, Incubation, Co-Immunoprecipitation Assay, Western Blot

Journal: PLOS Pathogens

Article Title: TRIM21 restricts influenza A virus replication by ubiquitination-dependent degradation of M1

doi: 10.1371/journal.ppat.1011472

Figure Lengend Snippet: (A) TRIM21 degrades H3/H5/H9 M1 rather than H1/H7 M1. Myc-tagged vector or Myc-tagged TRIM21 and Flag-tagged H1/H3/H5/H7/H9 M1 were co-transfected into HEK293T cells, then the cell lysates were detected using the indicated antibodies. The number under the gel band represent the relative protein concentration calculated using Image J software with the gray value of GAPDH as the control standard. (B) TRIM21 mediates H9N2 M1 degradation through the proteasomal pathway. HEK293T cells expressing Flag-tagged H9N2 M1 were transfected with Myc-tagged vector or Myc-tagged TRIM21 for 12 h, followed by treatment with MG132 (6.25μM, 12.5μM, 25μM) or chloroquine phosphate (12.5μM, 25μM, 50μM) for 6 h. Additionally, HEK293T cells expressing Flag-tagged H9N2 M1 R 95 K were co-transfected with Myc-tagged vector or Myc-tagged TRIM21 for 12 h, followed by treatment with MG132 (25μM) or chloroquine phosphate (50μM) for 6 h. The cell lysates were detected using the indicated antibodies. (C) TRIM21 ubiquitinates the M1 protein of H3N2, H5N1 and H9N2 viruses, but not those of H7N9 and PR8 viruses. Myc-tagged vector or the TRIM21 vector was co-transfected with Flag-GST-tagged H9N2 M1 or H7N9 M1, H5N1 M1, H3N2 M1, PR8 M1 and HA-tagged Ub-WT plasmids into HEK293T cells for 48 h, followed by treatment with 25μM MG132 for 6 h. The cell lysates were then subjected to immunoprecipitation and western blotting using the indicated antibodies. (D) TRIM21 promotes K48-linked polyubiquitination of H9N2 M1. Myc-tagged vector or the TRIM21 vector was co-transfected with Flag-GST-tagged H9N2 M1 and HA-tagged Ub-WT or Ub-K48 or Ub-K63 plasmids into HEK293T cells for 48 h, followed by treatment with 25μM MG132 for 6 h. The cell lysates were then subjected to immunoprecipitation. (E-H) R 95 and K 242 in H9N2 M1 were necessary for TRIM21 degradation. Myc-tagged vector or Myc-tagged TRIM21 and Flag-tagged H9N2 M1 WT or its mutants (T 37 A, R 95 K, S 242 N, K 242 N, R 95 K/K 242 N) were co-transfected into HEK293T cells, and the cell lysates were detected using the indicated antibodies (E and G). R 95 and K 242 in H9N2 M1 were required for TRIM21 ubiquitination. Myc-tagged vector and Myc-tagged TRIM21 WT plasmids were co-transfected with Flag-GST-tagged H9N2 M1 WT or its mutants (T 37 A, R 95 K, S 242 N, K 242 N, R 95 K/K 242 N) and HA-tagged Ub-WT plasmids in HEK293T cells for 48 h, followed by treatment with 25μM MG132 for 6 h. The cell lysates were then subjected to immunoprecipitation and western blotting using the indicated antibodies (F and H). (I) E3 ligase activity is indispensable for TRIM21-mediated degradation of H9N2 M1. Myc-tagged vector, Myc-tagged TRIM21 WT, its mutant C 16 A and TRIM21 deletion (ΔRING, ΔBB, ΔCC and ΔPRY/SPRY) and Flag-tagged H9N2 M1 were co-transfected into HEK293T cells for 12h. Then, the cell lysates were identified using the indicated antibodies. (J) E3 ligase activity was required to catalyze the ubiquitination of H9N2 M1. Myc-tagged vector, Myc-tagged TRIM21 WT, its mutant C 16 A and TRIM21 deletion (ΔRING, ΔBB, ΔCC and ΔPRY/SPRY) were co-transfected with Flag-gst-tagged H9N2 M1 and HA-tagged Ub-WT in HEK293T cells for 48 h, followed by treatment with 25μM MG132 for 6 h. Subsequently, the cell lysates were subjected to immunoprecipitation and western blotting using the indicated antibodies. Each experiment was independently performed with three biological repeats. All results are presented as means ± SD. *, p < 0.05; **, p < 0.01; ns, p > 0.05.

Article Snippet: Rabbit mAbs against TRIM21 (12108-1-AP) was purchased from Proteintech Group, Inc. (Manchester, UK).

Techniques: Plasmid Preparation, Transfection, Protein Concentration, Software, Control, Expressing, Immunoprecipitation, Western Blot, Ubiquitin Proteomics, Activity Assay, Mutagenesis

Journal: PLOS Pathogens

Article Title: TRIM21 restricts influenza A virus replication by ubiquitination-dependent degradation of M1

doi: 10.1371/journal.ppat.1011472

Figure Lengend Snippet: (A-D) TRIM21 inhibits H9N2 rather than PR8 IAV replication in TRIM21 overexpressing A549 cells. The TRIM21 overexpressing A549 cell lines and control cells were infected with H9N2 virus or PR8 virus at an MOI = 1.0 for 12 h and then the levels of protein (A), mRNA (B), vRNA (C) and the TCID 50 (D) were examined. The number under the gel band is the relative protein concentration calculated using Image J software with the gray value of GAPDH as the control standard. (E-H) TRIM21 increased H9N2 virus replication in TRIM21 -KO A549 cells. TRIM21 -KO A549 cells and control cells were infected with H9N2 virus or PR8 virus at an MOI = 1.0 for 12 h, and the levels of protein (E), mRNA (F), vRNA (G), and the TCID 50 (H) were examined. (I) One-step growth curves of H9N2 and mutant H9N2 (rH9N2). H9N2 and rH9N2 viruses were rescued in HEK293T cells, which were subsequently inoculated and propagated in 9-day-old SPF embryonated chicken eggs. Then, A549 cells were infected with an equal amount of allotonic fluid containing WT H9N2 or rH9N2 at 0.01 MOI for 24, 36, 48, and 60 h, respectively. The cell supernatant was harvested at the indicated time points, and the TCID 50 was calculated as described in the Materials and Methods. (J-K) TRIM21 restricted H9N2 replication but not rH9N2 in TRIM21 overexpressing A549 cell lines. TRIM21 overexpressing, TRIM21 -KO, and control A549 cells were infected with H9N2 or rH9N2 at an MOI = 1.0 for 12 h and then the viral titers were detected using TCID 50. *, p < 0.05; **, p < 0.01; ns, p > 0.05. Each experiment was independently performed with three biological repeats. All results are presented as means ± SD.

Article Snippet: Rabbit mAbs against TRIM21 (12108-1-AP) was purchased from Proteintech Group, Inc. (Manchester, UK).

Techniques: Control, Infection, Virus, Protein Concentration, Software, Mutagenesis

Journal: PLOS Pathogens

Article Title: TRIM21 restricts influenza A virus replication by ubiquitination-dependent degradation of M1

doi: 10.1371/journal.ppat.1011472

Figure Lengend Snippet: (A) One-step growth curve of WT and mutant H9N2 viruses. The WT and recombinant viruses were rescued in HEK293T cells and propagated in 9-day-old SPF embryonated chicken eggs. Then, A549 cells were infected with an equal amount of allotonic fluid containing WT and four mutant H9N2 viruses at 0.01 MOI for 24, 36, 48, and 60 h, respectively. The cell supernatant was harvested, and the TCID 50 was calculated as described in the Materials and Methods. (B-E) TRIM21 restricted the replication of WT and all mutant H9N2 viruses, except R 95 K virus and K 242 N virus, in A549 cells. TRIM21 -overexpressing, TRIM21 -KO, and control A549 cells were infected with H9N2 WT and mutants H9N2 M1-T 37 A, H9N2 M1-R 95 K, H9N2 M1-S 224 N, and H9N2 M1-K 242 N at an MOI = 1.0 for 12 h and then the levels of protein (B and C) and the TCID 50 (D and E) were examined. (F) the ubiquitination of the H9N2 WT and four mutant viruses except for R 95 K and K 242 N virus were markedly promoted by TRIM21. Myc-tagged vector or Myc-tagged TRIM21 and HA-tagged Ub-WT were co-transfected into HEK293T cells. After 24h, the cells were infected with H9N2 WT and mutant IAV (H9N2 M1-T 37 A, H9N2 M1-R 95 K, H9N2 M1-S 224 N, H9N2 M1-K 242 N) at an MOI = 1.0 for 12h. The cells were then treated with 25μM MG132 for 6 h before harvest, cell lysates were subjected to IP with anti-M1 and the precipitation was analyzed by western blot with the indicated antibodies. (G-J) The RING domain of TRIM21 is critical for inhibiting H9N2 virus replication. HEK293T cells were transfected with Myc-tagged vector or Myc-tagged TRIM21 WT, C 16 A mutant and ΔRING-deleted TRIM21 plasmids for 24 h, the cells were infected with H9N2 virus at an MOI = 1.0 for 12 h, and then the levels of viral protein (G), mRNA (H), vRNA (I), and the TCID 50 (J) were examined. Each experiment was independently performed with three biological repeats. All results are presented as means ± SD. *, p < 0.05; **, p < 0.01; ns, p > 0.05.

Article Snippet: Rabbit mAbs against TRIM21 (12108-1-AP) was purchased from Proteintech Group, Inc. (Manchester, UK).

Techniques: Mutagenesis, Recombinant, Infection, Virus, Control, Ubiquitin Proteomics, Plasmid Preparation, Transfection, Western Blot

Journal: PLOS Pathogens

Article Title: TRIM21 restricts influenza A virus replication by ubiquitination-dependent degradation of M1

doi: 10.1371/journal.ppat.1011472

Figure Lengend Snippet: The 8-week-old C57BL/6 mice (13 per group) were intranasally infected with WT H9N2 virus, H9N2 mutant viruses R 95 K, K 242 N, or R 95 K/K 242 N at a dose of 10 7.0 TCID 50 H9N2 virus, respectively. In another experiment, the 3-week-old C57BL/6 mice (thirteen per group) were intranasally infected with 10 11.0 TCID 50 of AAV6 with the TRIM21-shRNA (shTRIM21) or scrambled shRNA control (shCtrl). Four weeks after infection, the treated mice were intranasally infected with H9N2 (10 7.0 TCID 50 ). Three mice per group were euthanized on day 6 post-infection to check for lesions and virus replication in the lungs. The remaining mice (10 per group) were monitored until day 14. Mice with a weight loss of more than 30% of their initial body weight were euthanized and recorded as dead. (A and B) Curves of body weight (A) and survival (B) in mice (n = 10 mice) from 0 to 14 days after infection with WT H9N2 virus or H9N2 mutant viruses R 95 K, K 242 N and R 95 K/K 242 N. (C) Gross and histopathological lesions in the lung at 6 day after infection. (D) The lesion area was measured as a percentage and μm 2 of the total lung area in (C). (E-H) The lung of infected mice was harvested to detect the levels of viral proteins (E), mRNA level of M gene (F), viral RNA copies (G), and the TCID 50 (H). (I) Validation of shTRIM21 knockdown effect in mice. 3-week-old C57BL/6 mice were intranasally infected with shCtrl or shTRIM21 for four weeks, and TRIM21 expression in mouse lungs was detected by Western blot using the TRIM21 antibody. (J-K) Curves of body weight (J) and survival (K) in TRIM21 knocked down mice from 0 to 14 days after infection with WT H9N2 virus (n = 10 mice). (L) Gross and histopathological lesions in lungs of TRIM21 knocked down mice at 6 days after infection. (M) The lesion area was measured as a percentage and μm 2 of total lung area in (L). (N-Q) The lungs of TRIM21 knocked down mice with H9N2 infection were harvested to detect the levels of viral proteins (N), mRNA level of M gene (O), viral RNA copies (P), and the TCID 50 (Q). Each experiment was independently performed with three biological repeats. All results are presented as means ± SD.*, p < 0.05; **, p < 0.01; ns, p > 0.05. The photo by Lulu Lin.

Article Snippet: Rabbit mAbs against TRIM21 (12108-1-AP) was purchased from Proteintech Group, Inc. (Manchester, UK).

Techniques: Infection, Virus, Mutagenesis, shRNA, Control, Biomarker Discovery, Knockdown, Expressing, Western Blot

Journal: bioRxiv

Article Title: ASFV early protein p30 suppresses antiviral type I IFN induction by targeting TRIM21 and RIG-I like receptor signaling adaptor MAVS

doi: 10.64898/2026.03.26.714469

Figure Lengend Snippet: Interactions exist between ASFV p30, TRIM21 and MAVS. ( A ) 293T cells were co-transfected with GFP-MAVS and Flag-TRIM21 for 24 h. Immunoprecipitation was performed using the rabbit anti-GFP mAb to confirm the interaction between exogenous MAVS and TRIM21. ( B ) 293T cells were co-transfected with mCherry-MAVS, Flag-TRIM21, and p30-GFP for 24 h. Immunoprecipitation was performed using rabbit mCherry pAb to confirm the interactions between exogenous MAVS, TRIM21 and p30. ( C ) MA104 cells were infected with ASFV for 72 h. Immunoprecipitation were performed using rabbit anti-MAVS mAb to confirm the interactions between endogenous MAVS, TRIM21 and p30. ( D ) The purified p30, TRIM21 and MAVS recombinant proteins were confirmed by SDS-PAGE and labeled with red boxes. ( E-G ) The interactions between purified proteins p30 and TRIM21, p30 and MAVS, and MAVS and TRIM21 verified through immunoprecipitations.

Article Snippet: The rabbit anti-TRIM21 mAb, mouse anti-TRIM21 mAb, rabbit anti-MAVS mAb, rabbit anti-IRF3 mAb, rabbit anti-Flag mAb, rabbit anti-HA mAb and rabbit mCherry pAb were all from ProteinTech (Wuhan, China).

Techniques: Transfection, Immunoprecipitation, Infection, Purification, Recombinant, SDS Page, Labeling

Journal: bioRxiv

Article Title: ASFV early protein p30 suppresses antiviral type I IFN induction by targeting TRIM21 and RIG-I like receptor signaling adaptor MAVS

doi: 10.64898/2026.03.26.714469

Figure Lengend Snippet: ASFV p30 inhibits TRIM21 upregulated MAVS signaling. ( A ) 293T cells were co-transfected mCherry-MAVS, Flag-TRIM21 and p30-GFP, as indicated, for 24 h. The downstream ISG56 and IFN-β mRNA levels activated by MAVS in the presence of TRIM21 were analyzed using RT-qPCR. ( B ) 3D4/21 cells were co-transfected with Flag-TRIM21 and p30-GFP, as indicated, for 12 h, and stimulated by transfection of poly (I:C) for 12 h. The downstream ISG56 and IFN-β mRNA levels activated by poly (I:C) in the presence of TRIM21 were analyzed using RT-qPCR. ( C ) 293T cells were co-transfected with mCherry-MAVS, Flag-TRIM21 and p30-GFP, plus ISRE Fluc and Rluc reporters, as indicated, for 24 h, and ISRE promoter activity was measured via dual luciferase reporter assay. ( D and E ) 3D4/21 cells were co-transfected with Flag-TRIM21 and p30-GFP for 12 h, as indicated. Cells were infected with VSV (D) or stimulated with transfection of poly (I:C) (E), followed by Western blotting for detection of cell signaling activation. ( F ) PAMs were transfected with p30 siRNA or control siRNA for 24 h, stimulated with transfection of poly (I:C) for 6 h, and then infected with ASFV for 48 h. The cell signaling and ASFV replication were detected by Western blotting.

Article Snippet: The rabbit anti-TRIM21 mAb, mouse anti-TRIM21 mAb, rabbit anti-MAVS mAb, rabbit anti-IRF3 mAb, rabbit anti-Flag mAb, rabbit anti-HA mAb and rabbit mCherry pAb were all from ProteinTech (Wuhan, China).

Techniques: Transfection, Quantitative RT-PCR, Activity Assay, Luciferase, Reporter Assay, Infection, Western Blot, Activation Assay, Control

Journal: bioRxiv

Article Title: ASFV early protein p30 suppresses antiviral type I IFN induction by targeting TRIM21 and RIG-I like receptor signaling adaptor MAVS

doi: 10.64898/2026.03.26.714469

Figure Lengend Snippet: ASFV p30 inhibits TRIM21 mediated ubiquitination of MAVS K27 ubiquitination. ( A ) 293T cells were co-transfected with Flag-TRIM21, mCherry-MAVS and HA-Ub for 24 h and then treated with MG-132 (10 μm) for 6 h. ( B ) 293T cells were co-transfected with Flag-TRIM21, mCherry-MAVS, p30-GFP, and HA-Ub for 24 h and then treated with MG-132 (10 μm) for 6 h. Cell samples were collected and immunoprecipitation were used for analysis of MAVS ubiquitination. ( C ) 293T cells were co-transfected with Flag-TRIM21, mCherry-MAVS and HA-Ub-K27 for 24 h and then treated with MG-132 (10 μm) for 6 h. ( D ) 293T cells were co-transfected with Flag-TRIM21, mCherry-MAVS, p30-myc, and HA-Ub-K27 for 24 h and then treated with MG-132 (10 μm) for 6 h. Cell samples were collected and immunoprecipitation were used for analysis of MAVS K27 ubiquitination.

Article Snippet: The rabbit anti-TRIM21 mAb, mouse anti-TRIM21 mAb, rabbit anti-MAVS mAb, rabbit anti-IRF3 mAb, rabbit anti-Flag mAb, rabbit anti-HA mAb and rabbit mCherry pAb were all from ProteinTech (Wuhan, China).

Techniques: Ubiquitin Proteomics, Transfection, Immunoprecipitation

Journal: Viruses

Article Title: Porcine TRIM21 Enhances Porcine Circovirus 2 Infection and Host Immune Responses, But Inhibits Apoptosis of PCV2-Infected Cells

doi: 10.3390/v14010156

Figure Lengend Snippet: Porcine TRIM21 decreases cellular apoptosis in PCV2-infected cells. ( A ) p53. PK-15 cells were infected with PCV2 for 72 h, followed by Western blot. ( B ) Caspase 3, 8, and 9 activities. PK-15 cells (control cells, Con) and TRIM21-OE cells were infected with PCV2 for 72 h, followed by caspases activities analysis using the Caspase (3, 8, and 9) Activity Assay Kit. * p < 0.05; **** p < 0.0001. ( C ) Bcl-2. PK-15 cells were infected with PCV2 for 72 h, followed by Western blot. Mouse anti-P53 monoclonal antibody (1:1000), mouse anti-Bcl-2 polyclonal antibody (1:1000), and mouse anti-β-actin antibody (1:2000) were used as primary antibody, and HRP-labeled goat anti-mouse IgG (H + L, 1:2000) was used as the secondary antibody. Unprocessed original images can be found in .

Article Snippet: TRIM21 rabbit polyclonal antibody, mouse P53 monoclonal antibody, and mouse Bcl-2 polyclonal antibodies were purchased from Proteintech (Wuhan, China).

Techniques: Infection, Western Blot, Control, Activity Assay, Labeling

Journal: Virus research

Article Title: Systems genetics of influenza A virus-infected mice identifies TRIM21 as a critical regulator of pulmonary innate immune response.

doi: 10.1016/j.virusres.2024.199335

Figure Lengend Snippet: Fig. 8. Impact of TRIM21 depletion on expression of ISG proteins and IFN production in IAV-infected human lung adenocarcinoma epithelial cells. (A) Expression of TRIM21, IFIT1/ISG56, DDX58/RIG-I, IFIH1/MDA5, Vinculin (loading control), and IAV NP proteins in control A549 cells and TRIM21-knockdown cells (A549-shT21), mock-infected or infected by IAV 1–126 TX/98 (MOI = 2) for the indicated times. Data are representative of three independent experiments. Note that at 17 h.p.i., a breakdown product of IFIH1 protein (marked by diamond) was also detected and more so in shT21 cells (lane 6) than in control A549 (lane 3). Numbers in purple color under each target blot denote relative expression of the protein of interest after normalization to Vinculin loading control analyzed by Image Studio™ (LI-COR biosciences). (B) IFN production, as measured by antiviral activity present in culture supernatants under the indicated conditions. Data are compiled from two independent experiments and expressed as units/ml. Student’s T-test was used to compare the indicated groups for the presence of statistical significance (* denotes p<0.05). A representative crystal violet staining of the plaque-reduction assay cell plate from two independent experiments is shown in (C). Mock, culture supernatant of mock-infected cells; IAV 20 h, culture supernatant of cells infected by IAV 1–126 TX/98 (MOI=3) for 20 h.

Article Snippet: For immunodetection, we used the following monoclonal (mAb) and polyclonal (pAb) antibodies: mouse anti-IAV NP mAb 5/1 (Varich et al., 2009); Rabbit anti-TRIM21 mAb (Cell Signaling Technology); rabbit anti-IFIT1/ISG56 pAb (Wang et al., 2009); rabbit anti-IFIH1/MDA5 pAb (Proteintech); rabbit anti-DDX58/RIG-I pAb (Enzo Life Sciences); mouse anti-Vinculin mAb (Proteintech); and appropriate IRDye® secondary antibodies - goat anti-mouse IgG IRDye® 680RD or goat anti-rabbit IgG IRDye® 800CW (both from LI-COR Biosciences).

Techniques: Expressing, Infection, Control, Knockdown, Activity Assay, Staining