Journal: International Journal of Biological Sciences

Article Title: HPV E7 inhibits cell pyroptosis by promoting TRIM21-mediated degradation and ubiquitination of the IFI16 inflammasome

doi: 10.7150/ijbs.50074

Figure Lengend Snippet: HPV E7 interacts with IFI16 and the E3 ligaseTRIM21. (A) Mass spectrum data of TRIM21 among HPV 11E7-interacting proteins identified by mass spectrometry. (B) Immunoblot analysis of HaCaT cells stably expressing HPV 11E7 transfected with poly(dA:dT) for the indicated times, followed by immunoprecipitation with anti-FlagM2 beads. (C) Immunoblot analysis of HeLa cells transfected with poly(dA:dT) for the indicated times, followed by immunoprecipitation with IFI16, HPV 18E7, TRIM21 or immunoglobulin G (IgG)-conjugated magnetic beads. (D) Coimmunoprecipitation and immunoblot analysis of 293T cells cotransfected for 36 hr with Flag-IFI16 plus Myc-TRIM21 or Myc-TRIM21 mutant vectors, followed by immunoprecipitation with anti-Flag-M2 beads. (E) Coimmunoprecipitation and immunoblot analysis of 293T cells cotransfected for 36 hr with Flag-HPV 18E7 plus Myc-TRIM21 or Myc-TRIM21 mutant vectors, followed by immunoprecipitation with anti-FlagM2 beads. (F) Coimmunoprecipitation and immunoblot analysis of 293T cells cotransfected for 36 hr with Myc-TRIM21 and Flag-IFI16 or Flag-IFI16 mutant vectors, followed by immunoprecipitation with anti-FlagM2 beads.

Article Snippet: Antibodies specific for IFI16 (sc-8023, 1:1000), TRIM21 (sc25351, 1:1000), ubiquitin (sc-471120, 1:1000), and GFP (sc-390394) were from Santa Cruz, Inc. Antibodies against Flag (M20008M), Myc (M20002M), mouse IgG (B30010M), and rabbit IgG (B30011S) were obtained from Abmart.

Techniques: Mass Spectrometry, Western Blot, Stable Transfection, Expressing, Transfection, Immunoprecipitation, Magnetic Beads, Mutagenesis

Journal: International Journal of Biological Sciences

Article Title: HPV E7 inhibits cell pyroptosis by promoting TRIM21-mediated degradation and ubiquitination of the IFI16 inflammasome

doi: 10.7150/ijbs.50074

Figure Lengend Snippet: HPV E7 promotes the K33-linked ubiquitination and degradation of IFI16 mediated by TRIM21. (A) Immunoblot analysis of lysates in control HeLa cells or TRIM21 knockout stable HeLa cells transfected with poly(dA:dT) for the indicated times. (B) Immunoblot analysis of IFI16 in the lysates of TRIM21 knockout stable HeLa cells treated with CHX (40 μg/ml) for the indicated number of hours after transfection with poly(dA:dT) for 1 hr. (C) Immunoblot analysis of 293T cells cotransfected with the Myc-TRIM21 and Flag-IFI16 vectors for 36 hr. (D) Immunoblot analysis of 293T cells cotransfected with the Myc-TRIM21 and Flag-IFI16 vectors with or without MG132 treatment. (E) Immunoblot analysis of 293T cells cotransfected with the Myc-TRIM21 and Flag-IFI16 vectors treated with CHX (40 μg/ml) for the indicated number of hours. (F) Immunoblot analysis of 293T cells cotransfected with Myc-TRIM21and the Flag-IFI16 or GFP-HPV 18E7 vector for 36 hr. (G) Immunoblot analysis of ubiquitinated IFI16 in control HeLa cells or TRIM21 knockout stable HeLa cells transfected with poly(dA:dT) for the indicated times and treated with MG132 for 6 hr before cell harvest. (H) Immunoblot analysis of ubiquitinated IFI16 in 293T cells cotransfected with Myc-TRIM21and the Flag-IFI16 or HA-ub vector for 36 hr with or without MG132 treatment for 6 hr before cell harvest. (I) Immunoblot analysis of ubiquitinated IFI16 in 293T cells transfected with combinations of the Myc-TRIM21, Flag-IFI16, HA-ub, and GFP-HPV 18E7 vectors for 36 hr and treated with MG132 for 6 hr before cell harvest. (J) Immunoblot analysis of ubiquitinated IFI16 in 293T cells transfected with combinations of the Myc-TRIM21, Flag-IFI16, HA-ub, and HA-ub mutant (each of the Lys residues were replaced by an Arg residue) vectors for 36 hr and treated with MG132 for 6 hr before cell harvest. (K) Immunoblot analysis of ubiquitinated IFI16 in 293T cells transfected with combinations of the Myc-TRIM21, Flag-IFI16, HA-ub, HA-ub mutant (all Lys residues but one was replaced with Arg residues) vectors for 36 hr and treated with MG132 for 6 hr before cell harvest. Data are representative of at least three independent experiments.

Article Snippet: Antibodies specific for IFI16 (sc-8023, 1:1000), TRIM21 (sc25351, 1:1000), ubiquitin (sc-471120, 1:1000), and GFP (sc-390394) were from Santa Cruz, Inc. Antibodies against Flag (M20008M), Myc (M20002M), mouse IgG (B30010M), and rabbit IgG (B30011S) were obtained from Abmart.

Techniques: Ubiquitin Proteomics, Western Blot, Control, Knock-Out, Transfection, Plasmid Preparation, Mutagenesis, Residue

Journal: International Journal of Biological Sciences

Article Title: HPV E7 inhibits cell pyroptosis by promoting TRIM21-mediated degradation and ubiquitination of the IFI16 inflammasome

doi: 10.7150/ijbs.50074

Figure Lengend Snippet: TRIM21 downregulates cell pyroptosis induced by poly(dA:dT). (A) Microscopy imaging of cell death in control HeLa cells or TRIM21 knockout stable HeLa cells transfected with poly(dA:dT) for 18 hr. (B) Flow cytometry analysis of propidium iodide-positive control HeLa cells or TRIM21 knockout stable HeLa cells transfected with poly(dA:dT) for 16 hr. (C) Cell viability assay in control HeLa cells or TRIM21 knockout stable HeLa cells transfected with poly(dA:dT) for the indicated times. (D) LDH assay in control HeLa cells or TRIM21 knockout stable HeLa cells transfected with poly(dA:dT) for the indicated times. (E) Immunoblot analysis of GSDMD and caspase-1 in the lysates of control HeLa cells or TRIM21 knockout stable HeLa cells transfected with poly(dA:dT) for 24 hr. (F) ELISA analysis of IL-18 and IL-1β in control HeLa cells or knockout stable HeLa cells transfected with poly(dA:dT) for 24 hr. Data are presented as mean ± SD of duplicate samples and are representative of at least three independent experiments. P values are determined by two-tailed Student's t test. ** p < 0.01, *** p < 0.001.

Article Snippet: Antibodies specific for IFI16 (sc-8023, 1:1000), TRIM21 (sc25351, 1:1000), ubiquitin (sc-471120, 1:1000), and GFP (sc-390394) were from Santa Cruz, Inc. Antibodies against Flag (M20008M), Myc (M20002M), mouse IgG (B30010M), and rabbit IgG (B30011S) were obtained from Abmart.

Techniques: Microscopy, Imaging, Control, Knock-Out, Transfection, Flow Cytometry, Positive Control, Viability Assay, Lactate Dehydrogenase Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: HPV E7 inhibits cell pyroptosis by promoting TRIM21-mediated degradation and ubiquitination of the IFI16 inflammasome

doi: 10.7150/ijbs.50074

Figure Lengend Snippet: Schematic diagram showing that HPV E7 interacted with IFI16 and promoted the ubiquitin-mediated degradation of IFI16 by recruiting the E3 ligase TRIM21, resulting in the inhibition of cell pyroptosis during HPV infection.

Article Snippet: Antibodies specific for IFI16 (sc-8023, 1:1000), TRIM21 (sc25351, 1:1000), ubiquitin (sc-471120, 1:1000), and GFP (sc-390394) were from Santa Cruz, Inc. Antibodies against Flag (M20008M), Myc (M20002M), mouse IgG (B30010M), and rabbit IgG (B30011S) were obtained from Abmart.

Techniques: Ubiquitin Proteomics, Inhibition, 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 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 transfected with pcDNA3.1-ROP18 I -FLAG using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific) for 24 h. The cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) followed by Western blotting.

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 transfected with pcDNA3.1-ROP18 I -FLAG using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific) for 24 h. The cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) followed by Western blotting.

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 transfected with pcDNA3.1-ROP18 I -FLAG using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific) for 24 h. The cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) followed by Western blotting.

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 transfected with pcDNA3.1-ROP18 I -FLAG using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific) for 24 h. The cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) followed by Western blotting.

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 transfected with pcDNA3.1-ROP18 I -FLAG using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific) for 24 h. The cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) followed by Western blotting.

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 transfected with pcDNA3.1-ROP18 I -FLAG using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific) for 24 h. The cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) followed by Western blotting.

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 transfected with pcDNA3.1-ROP18 I -FLAG using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific) for 24 h. The cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) followed by Western blotting.

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 transfected with pcDNA3.1-ROP18 I -FLAG using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific) for 24 h. The cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) followed by Western blotting.

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 transfected with pcDNA3.1-ROP18 I -FLAG using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific) for 24 h. The cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) followed by Western blotting.

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 transfected with pcDNA3.1-ROP18 I -FLAG using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific) for 24 h. The cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) followed by Western blotting.

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

Journal: International Journal of Oncology

Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

doi: 10.3892/ijo.2015.3169

Figure Lengend Snippet: TRIM21 domain. (A) (Left) Vector map of the used pMK-RQ vector (Life Technologies), (right) amino acid sequence of TRIM21 and the sequences of an individual domains. (B) Western blot analysis after native 12.5% polyacrylamide gel. Detection of TRIM21 with Li-Cor 800 anti-goat, detection of LFG with Li-Cor-680 anti-rabbit. Sample 1a (PRY domain), sample 1b (SPRY domain), sample 2 (coiled-coil domain), sample 3 (B-box domain), sample 4 (RING domain), marker (M). The samples 2–4 show clearly visible bands of ~80 and 60 kDa. The bands at 80 kDa are larger but slightly less visible as the bands at the height of 60 kDa. In samples 1a and 1b no bands are visible.

Article Snippet: To identify the essential domain for the interaction between TRIM21 and LFG, different vectors, using pCMV6-AC-GFP vector (NM_003141; OriGene, Cambridge, UK) were cloned containing the sequences of TRIM21 with the deletion of an entire domain.

Techniques: Plasmid Preparation, Sequencing, Western Blot, Marker

Journal: International Journal of Oncology

Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

doi: 10.3892/ijo.2015.3169

Figure Lengend Snippet: Analysis of the interaction. Array analysis of the interaction of over 9,000 different proteins with LFG. Recombinant LFG protein (10 μM) was used and detected by specific first antibody (FAIM2; Santa Cruz Biotechnology, Inc.) and fluorescence labeled second antibody (Alexa Fluor 546). The display window shows the signal stimulated by the interaction of LFG with TRIM21 (white signal).

Article Snippet: To identify the essential domain for the interaction between TRIM21 and LFG, different vectors, using pCMV6-AC-GFP vector (NM_003141; OriGene, Cambridge, UK) were cloned containing the sequences of TRIM21 with the deletion of an entire domain.

Techniques: Recombinant, Fluorescence, Labeling

Journal: International Journal of Oncology

Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

doi: 10.3892/ijo.2015.3169

Figure Lengend Snippet: Z-score of protein-protein interaction array-analysis.

Article Snippet: To identify the essential domain for the interaction between TRIM21 and LFG, different vectors, using pCMV6-AC-GFP vector (NM_003141; OriGene, Cambridge, UK) were cloned containing the sequences of TRIM21 with the deletion of an entire domain.

Techniques: Variant Assay, Activation Assay

Journal: International Journal of Oncology

Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

doi: 10.3892/ijo.2015.3169

Figure Lengend Snippet: Co-immunoprecipitation. (A) Native western blot analysis after co-immunoprecipitation of LFG and TRIM21. LFG was specifically isolated from the cell lysate of MDA-MB-231 after transfection with vectors coding for LFG and TRIM21 (24 h) by use of antibody coated magnetic beads (Dynabeads; Invitrogen). Detection of LFG (FAIM2) and TRIM21 (Ssa1/2) (both from Santa Cruz Biotechnology, Inc.) by specific first antibodies and fluorescence labeled second antibodies. LFG is visible as a green signal (Alexa Fluor 546), TRIM21 is visible as a red signal (Alexa Fluor 488), and combined signals are visible as a yellow signal. (B) Analysis of LFG and TRIM21 protein in the MDA-MB-231 breast cancer cells 24 h after transfection with vectors coding for LFG and TRIM21, by specific first and fluorescence labeled second antibodies. a, Green signal for LFG; b, red signal for TRIM21; c, blue signal for DAPI-stained core; d, overlay of the single signals with yellow signals in places where green and red signals appear in the same spot.

Article Snippet: To identify the essential domain for the interaction between TRIM21 and LFG, different vectors, using pCMV6-AC-GFP vector (NM_003141; OriGene, Cambridge, UK) were cloned containing the sequences of TRIM21 with the deletion of an entire domain.

Techniques: Immunoprecipitation, Western Blot, Isolation, Transfection, Magnetic Beads, Fluorescence, Labeling, Staining

Journal: International Journal of Oncology

Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

doi: 10.3892/ijo.2015.3169

Figure Lengend Snippet: Trim21 and LFG expression. (A) Results of real-time PCR-analysis of Trim21-expression after 24 h under different culture conditions. Mock, Untreated MDA-MB-231 as negative control, Trim21, 2.0 μl recombinant human Trim21 protein added to culture medium; siControl, adenoviral transfection of MDA-MB-231 with empty vector; siLFG, adenoviral transfection of MDA-MB-231 with vector coding for siRNA against LFG. (B) Expression analysis of LFG protein under different culture conditions by western blot analysis using 25 μg of total protein. a, Adenoviral transfection of MDA-MB-231 with vector coding for siRNA against LFG (24 h). b, Adenoviral transfection of MDA-MB-231 with empty vector. c, Untreated MDA-MB-231 as negative control. d, MDA-MB231 cultivated with 2.0 μg recombinant Trim21-protein in culture medium.

Article Snippet: To identify the essential domain for the interaction between TRIM21 and LFG, different vectors, using pCMV6-AC-GFP vector (NM_003141; OriGene, Cambridge, UK) were cloned containing the sequences of TRIM21 with the deletion of an entire domain.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Negative Control, Recombinant, Transfection, Plasmid Preparation, Western Blot

Journal: International Journal of Oncology

Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

doi: 10.3892/ijo.2015.3169

Figure Lengend Snippet: Caspase-3-activity and cell cycle-analysis. (A) Analysis of caspase-3-activity in MDA-MB-231 cells. Cultivation for 48 h under different conditions and addition of 100 ng agonistic anti-Fas (clone CH11; Abcam). Measurement of caspase-3-activity after 24 h. TRIM21, addition of 1.0 μg/ml recombinant human TRIM21-protein every 24 h. Control, untreated MDA-MB-231 cultivated for 48 h. Detection of LFG, TRIM21 and actin by specific first antibodies and fluorescence labeled second antibodies. (B) Plots of cell cycle-analysis of MDA-MB-231 after 24 h cultivation in presence of different concentration of recombinant human TRIM21-protein after staining with propidium iodide. a-1, Cultivation of MDA-MB-231 without addition of TRIM21-recombinant protein. b-1, Addition of 0.25 μg/ml TRIM21-recombinant protein to the culture medium. c-1, Addition of 0.5 μg/ml TRIM21-recombinant protein to the culture medium. d-1, Addition of 0.75 μg/ml TRIM21-recombinant protein to the culture medium. e-1, Addition of 1.0 μg/ml TRIM21-recombinant protein to the culture medium.

Article Snippet: To identify the essential domain for the interaction between TRIM21 and LFG, different vectors, using pCMV6-AC-GFP vector (NM_003141; OriGene, Cambridge, UK) were cloned containing the sequences of TRIM21 with the deletion of an entire domain.

Techniques: Activity Assay, Cell Cycle Assay, Recombinant, Fluorescence, Labeling, Concentration Assay, Staining

Journal: International Journal of Oncology

Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

doi: 10.3892/ijo.2015.3169

Figure Lengend Snippet: Arrays. (A) Tissue samples of breast tumors having different degrees of malignity (US Biomax, Inc.) (a and b). Specific antibody against Trim21 and fluorescence labeled second antibody (Li-Cor-800), and staining of nucleic acid with Syto-60. Detection was carried out using Odyssey (Li-Cor Biosciences). (B) Bar chart of the fluorescence intensity of the tissue samples, degrees of malignity (IIa, IIb, IIIa, IIIb and IV); * P<0.05 vs. control (NAT). NAT, native tissue sample. Benign, benign breast tumor. Inflammation, inflamed tissue sample.

Article Snippet: To identify the essential domain for the interaction between TRIM21 and LFG, different vectors, using pCMV6-AC-GFP vector (NM_003141; OriGene, Cambridge, UK) were cloned containing the sequences of TRIM21 with the deletion of an entire domain.

Techniques: Fluorescence, Labeling, Staining

Journal: International Journal of Oncology

Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

doi: 10.3892/ijo.2015.3169

Figure Lengend Snippet: NF-κB array. Bar chart of gene expression of common NF-κB regulated genes after real-time PCR-analysis. Trim21, MDA-MB-231 cultivated with 2.0 μg/ml recombinant human Trim21-protein for 24 h. Control, MDA-MB-231 cells cultivated for 24 h under standard conditions.

Article Snippet: To identify the essential domain for the interaction between TRIM21 and LFG, different vectors, using pCMV6-AC-GFP vector (NM_003141; OriGene, Cambridge, UK) were cloned containing the sequences of TRIM21 with the deletion of an entire domain.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Recombinant

Journal: International Journal of Oncology

Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

doi: 10.3892/ijo.2015.3169

Figure Lengend Snippet: TRIM21 domain. (A) (Left) Vector map of the used pMK-RQ vector (Life Technologies), (right) amino acid sequence of TRIM21 and the sequences of an individual domains. (B) Western blot analysis after native 12.5% polyacrylamide gel. Detection of TRIM21 with Li-Cor 800 anti-goat, detection of LFG with Li-Cor-680 anti-rabbit. Sample 1a (PRY domain), sample 1b (SPRY domain), sample 2 (coiled-coil domain), sample 3 (B-box domain), sample 4 (RING domain), marker (M). The samples 2–4 show clearly visible bands of ~80 and 60 kDa. The bands at 80 kDa are larger but slightly less visible as the bands at the height of 60 kDa. In samples 1a and 1b no bands are visible.

Article Snippet: Briefly, 1×10 4 MDA-MB-231 breast cancer cells were seeded per well of a 96-well plate, and incubated with 1 μg TRIM21 human recombinant protein (ProSpec; Ness Ziona, Tel Aviv, Israel) every 24 h. After 48 h, 100 ng of agonistic anti-Fas (clone CH11; Abcam) were added and after 24 h, incubated cells were mixed with 100 μl of Apo-One homogeneous caspase-3/7 reagent.

Techniques: Plasmid Preparation, Sequencing, Western Blot, Marker

Journal: International Journal of Oncology

Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

doi: 10.3892/ijo.2015.3169

Figure Lengend Snippet: Analysis of the interaction. Array analysis of the interaction of over 9,000 different proteins with LFG. Recombinant LFG protein (10 μM) was used and detected by specific first antibody (FAIM2; Santa Cruz Biotechnology, Inc.) and fluorescence labeled second antibody (Alexa Fluor 546). The display window shows the signal stimulated by the interaction of LFG with TRIM21 (white signal).

Article Snippet: Briefly, 1×10 4 MDA-MB-231 breast cancer cells were seeded per well of a 96-well plate, and incubated with 1 μg TRIM21 human recombinant protein (ProSpec; Ness Ziona, Tel Aviv, Israel) every 24 h. After 48 h, 100 ng of agonistic anti-Fas (clone CH11; Abcam) were added and after 24 h, incubated cells were mixed with 100 μl of Apo-One homogeneous caspase-3/7 reagent.

Techniques: Recombinant, Fluorescence, Labeling

Journal: International Journal of Oncology

Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

doi: 10.3892/ijo.2015.3169

Figure Lengend Snippet: Z-score of protein-protein interaction array-analysis.

Article Snippet: Briefly, 1×10 4 MDA-MB-231 breast cancer cells were seeded per well of a 96-well plate, and incubated with 1 μg TRIM21 human recombinant protein (ProSpec; Ness Ziona, Tel Aviv, Israel) every 24 h. After 48 h, 100 ng of agonistic anti-Fas (clone CH11; Abcam) were added and after 24 h, incubated cells were mixed with 100 μl of Apo-One homogeneous caspase-3/7 reagent.

Techniques: Variant Assay, Activation Assay

Journal: International Journal of Oncology

Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

doi: 10.3892/ijo.2015.3169

Figure Lengend Snippet: Co-immunoprecipitation. (A) Native western blot analysis after co-immunoprecipitation of LFG and TRIM21. LFG was specifically isolated from the cell lysate of MDA-MB-231 after transfection with vectors coding for LFG and TRIM21 (24 h) by use of antibody coated magnetic beads (Dynabeads; Invitrogen). Detection of LFG (FAIM2) and TRIM21 (Ssa1/2) (both from Santa Cruz Biotechnology, Inc.) by specific first antibodies and fluorescence labeled second antibodies. LFG is visible as a green signal (Alexa Fluor 546), TRIM21 is visible as a red signal (Alexa Fluor 488), and combined signals are visible as a yellow signal. (B) Analysis of LFG and TRIM21 protein in the MDA-MB-231 breast cancer cells 24 h after transfection with vectors coding for LFG and TRIM21, by specific first and fluorescence labeled second antibodies. a, Green signal for LFG; b, red signal for TRIM21; c, blue signal for DAPI-stained core; d, overlay of the single signals with yellow signals in places where green and red signals appear in the same spot.

Article Snippet: Briefly, 1×10 4 MDA-MB-231 breast cancer cells were seeded per well of a 96-well plate, and incubated with 1 μg TRIM21 human recombinant protein (ProSpec; Ness Ziona, Tel Aviv, Israel) every 24 h. After 48 h, 100 ng of agonistic anti-Fas (clone CH11; Abcam) were added and after 24 h, incubated cells were mixed with 100 μl of Apo-One homogeneous caspase-3/7 reagent.

Techniques: Immunoprecipitation, Western Blot, Isolation, Transfection, Magnetic Beads, Fluorescence, Labeling, Staining

Journal: International Journal of Oncology

Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

doi: 10.3892/ijo.2015.3169

Figure Lengend Snippet: Trim21 and LFG expression. (A) Results of real-time PCR-analysis of Trim21-expression after 24 h under different culture conditions. Mock, Untreated MDA-MB-231 as negative control, Trim21, 2.0 μl recombinant human Trim21 protein added to culture medium; siControl, adenoviral transfection of MDA-MB-231 with empty vector; siLFG, adenoviral transfection of MDA-MB-231 with vector coding for siRNA against LFG. (B) Expression analysis of LFG protein under different culture conditions by western blot analysis using 25 μg of total protein. a, Adenoviral transfection of MDA-MB-231 with vector coding for siRNA against LFG (24 h). b, Adenoviral transfection of MDA-MB-231 with empty vector. c, Untreated MDA-MB-231 as negative control. d, MDA-MB231 cultivated with 2.0 μg recombinant Trim21-protein in culture medium.

Article Snippet: Briefly, 1×10 4 MDA-MB-231 breast cancer cells were seeded per well of a 96-well plate, and incubated with 1 μg TRIM21 human recombinant protein (ProSpec; Ness Ziona, Tel Aviv, Israel) every 24 h. After 48 h, 100 ng of agonistic anti-Fas (clone CH11; Abcam) were added and after 24 h, incubated cells were mixed with 100 μl of Apo-One homogeneous caspase-3/7 reagent.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Negative Control, Recombinant, Transfection, Plasmid Preparation, Western Blot

Journal: International Journal of Oncology

Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

doi: 10.3892/ijo.2015.3169

Figure Lengend Snippet: Caspase-3-activity and cell cycle-analysis. (A) Analysis of caspase-3-activity in MDA-MB-231 cells. Cultivation for 48 h under different conditions and addition of 100 ng agonistic anti-Fas (clone CH11; Abcam). Measurement of caspase-3-activity after 24 h. TRIM21, addition of 1.0 μg/ml recombinant human TRIM21-protein every 24 h. Control, untreated MDA-MB-231 cultivated for 48 h. Detection of LFG, TRIM21 and actin by specific first antibodies and fluorescence labeled second antibodies. (B) Plots of cell cycle-analysis of MDA-MB-231 after 24 h cultivation in presence of different concentration of recombinant human TRIM21-protein after staining with propidium iodide. a-1, Cultivation of MDA-MB-231 without addition of TRIM21-recombinant protein. b-1, Addition of 0.25 μg/ml TRIM21-recombinant protein to the culture medium. c-1, Addition of 0.5 μg/ml TRIM21-recombinant protein to the culture medium. d-1, Addition of 0.75 μg/ml TRIM21-recombinant protein to the culture medium. e-1, Addition of 1.0 μg/ml TRIM21-recombinant protein to the culture medium.

Article Snippet: Briefly, 1×10 4 MDA-MB-231 breast cancer cells were seeded per well of a 96-well plate, and incubated with 1 μg TRIM21 human recombinant protein (ProSpec; Ness Ziona, Tel Aviv, Israel) every 24 h. After 48 h, 100 ng of agonistic anti-Fas (clone CH11; Abcam) were added and after 24 h, incubated cells were mixed with 100 μl of Apo-One homogeneous caspase-3/7 reagent.

Techniques: Activity Assay, Cell Cycle Assay, Recombinant, Control, Fluorescence, Labeling, Concentration Assay, Staining

Journal: International Journal of Oncology

Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

doi: 10.3892/ijo.2015.3169

Figure Lengend Snippet: Arrays. (A) Tissue samples of breast tumors having different degrees of malignity (US Biomax, Inc.) (a and b). Specific antibody against Trim21 and fluorescence labeled second antibody (Li-Cor-800), and staining of nucleic acid with Syto-60. Detection was carried out using Odyssey (Li-Cor Biosciences). (B) Bar chart of the fluorescence intensity of the tissue samples, degrees of malignity (IIa, IIb, IIIa, IIIb and IV); * P<0.05 vs. control (NAT). NAT, native tissue sample. Benign, benign breast tumor. Inflammation, inflamed tissue sample.

Article Snippet: Briefly, 1×10 4 MDA-MB-231 breast cancer cells were seeded per well of a 96-well plate, and incubated with 1 μg TRIM21 human recombinant protein (ProSpec; Ness Ziona, Tel Aviv, Israel) every 24 h. After 48 h, 100 ng of agonistic anti-Fas (clone CH11; Abcam) were added and after 24 h, incubated cells were mixed with 100 μl of Apo-One homogeneous caspase-3/7 reagent.

Techniques: Fluorescence, Labeling, Staining, Control

Journal: International Journal of Oncology

Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

doi: 10.3892/ijo.2015.3169

Figure Lengend Snippet: NF-κB array. Bar chart of gene expression of common NF-κB regulated genes after real-time PCR-analysis. Trim21, MDA-MB-231 cultivated with 2.0 μg/ml recombinant human Trim21-protein for 24 h. Control, MDA-MB-231 cells cultivated for 24 h under standard conditions.

Article Snippet: Briefly, 1×10 4 MDA-MB-231 breast cancer cells were seeded per well of a 96-well plate, and incubated with 1 μg TRIM21 human recombinant protein (ProSpec; Ness Ziona, Tel Aviv, Israel) every 24 h. After 48 h, 100 ng of agonistic anti-Fas (clone CH11; Abcam) were added and after 24 h, incubated cells were mixed with 100 μl of Apo-One homogeneous caspase-3/7 reagent.

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Recombinant, Control

Journal: JCI Insight

Article Title: Inflammation-induced TRIM21 represses hepatic steatosis by promoting the ubiquitination of lipogenic regulators

doi: 10.1172/jci.insight.164694

Figure Lengend Snippet: ( A ) Immunostaining using anti-TRIM21 in liver sections from Ad-shTrim21 or Ad-Ctrl C57BL/6N mice fed a NASH diet for 30 weeks. ( B and C ) Relative hepatic mRNA expression of Trim21 ( B ), and A1cf and Khk ( C ). ( D ) Western blot analysis of indicated proteins ( n = 3 mice per group). ( E – I ) Liver weight ( E ), ratios of liver/body weight ( F ), liver TG ( G ), H&E and Oil Red O staining of liver sections ( H ), and immunoblot analysis of indicated lipogenesis genes ( I ) ( n = 3 mice per group) from mice indicated as in A . ( J–N ) Endogenous ubiquitination of SREBP1 ( J ), ChREBP ( K ), ACC ( L ), FASN ( M ), and A1CF ( N ) from liver extracts from mice indicated as in A ( n = 2). ( O ) Relative mRNA expression of de novo lipogenesis and glycolysis genes from mice indicated as in A . Scale bars: 50 μm. n represents number of replicates, 1 mouse/replicate. In all statistical plots, data are expressed as the mean ± SD; **** P < 0.0001; *** P < 0.001; ** P < 0.01; * P < 0.05. Statistical analysis for B , C , E–G , and O were carried out by 1-way ANOVA with Sidak’s post hoc analysis, n = 6 mice per group.

Article Snippet: The plasmids included: pLenti-msA1CF-Myc–expressing mouse A1CF (NM_001081074) tagged with Myc (MR224176L1), pRS-ms-shTRIM21 (TR514859), and the PCMV6-huTRIM21-expressing human TRIM21 cDNA (NM_003141) tagged by Myc (RC202088), provided by Origene.

Techniques: Immunostaining, Expressing, Western Blot, Staining

Journal: Viruses

Article Title: The E3 Ubiquitin Ligase TRIM21 Promotes HBV DNA Polymerase Degradation

doi: 10.3390/v12030346

Figure Lengend Snippet: The primers and oligonucleotides used in this work.

Article Snippet: The full-length sequences of human TRIM21 (Tianjin Saier Biotechnology, SRCL13641) cDNA was obtained by RT-PCR and cloned into the pcDNA3 vector with FLAG, Myc or HA tag between the EcoRI and XhoI sites, and the deletion fragments and point mutants were cloned the same as above.

Techniques: Sequencing, Ubiquitin Proteomics

Journal: Viruses

Article Title: The E3 Ubiquitin Ligase TRIM21 Promotes HBV DNA Polymerase Degradation

doi: 10.3390/v12030346

Figure Lengend Snippet: TRIM21 interacts with HBV DNA Pol. ( A ) Huh7 cells were transfected with FLAG-HBV DNA Pol and vector control, and FLAG affinity beads were used to precipitate all proteins that might interact with HBV DNA Pol. The interacting proteins of HBV DNA Pol were screened by mass spectrometry. Some potential interacting partners of HBV DNA Pol are listed. Red marks the target protein, blue marks proteins that have been reported to interact with HBV DNA Pol. Data are representative of two independent experiments. ( B ) The FLAG-HBV DNA Pol expression plasmid was transfected into Huh7 cells. After 36h, a co-IP assay was carried out with anti-FLAG antibody and control IgG antibody. HBV DNA Pol was then detected with anti-FLAG antibody, and anti-TRIM21 antibody was used to detect the endogenous expression of TRIM21 in Huh7 cells by Western blot. ( C ) Huh7 cells were cotransfected with FLAG-HBV DNA Pol and HA-TRIM21, and co-IP was performed as described in ( B ) to detect the expression of HBV DNA Pol and TRIM21. ( D ) Huh7 cells were transfected as described in ( C ), co-IP was performed with anti-HA antibody, and HBV DNA Pol and HA-TRIM21 expression were detected by Western blot. ( E ) GST-HBV DNA Pol and HA-TRIM21 were transfected into Huh7 cells, 36h after transfection, GST beads were used to pull down GST-Pol with its interacting proteins. HBV DNA Pol was detected with anti-GST antibody, and anti-HA antibody was used to detect the expression of TRIM21. ( F ) FLAG-HBV DNA Pol and HA-TRIM21 were transfected into Huh7 cells; 36h after transfection, the mouse anti-FLAG TRITC-conjugated monoclonal antibody and rabbit anti-HA FITC-conjugated polyclonal antibody were used to stain the cells, and the DAPI were used to stain the nuclei. Then, confocal microscopy images were collected and analyzed for the colocalization of HBV DNA Pol and TRIM21. The scale is 10 μm. Data are representative of three independent experiments with three replicates each.

Article Snippet: The full-length sequences of human TRIM21 (Tianjin Saier Biotechnology, SRCL13641) cDNA was obtained by RT-PCR and cloned into the pcDNA3 vector with FLAG, Myc or HA tag between the EcoRI and XhoI sites, and the deletion fragments and point mutants were cloned the same as above.

Techniques: Transfection, Plasmid Preparation, Control, Mass Spectrometry, Expressing, Co-Immunoprecipitation Assay, Western Blot, Staining, Confocal Microscopy

Journal: Viruses

Article Title: The E3 Ubiquitin Ligase TRIM21 Promotes HBV DNA Polymerase Degradation

doi: 10.3390/v12030346

Figure Lengend Snippet: TRIM21 interacts with the TP domain of HBV DNA Pol via its SPRY domain. ( A ) Schematic map of the deletion mutants of HBV DNA Pol. ( B ) HA-TRIM21 was cotransfected with FLAG-HBV DNA Pol, ΔTP, ΔRT or ΔRH. Anti-HA antibody was used for immunoprecipitation, and anti-FLAG antibody was used to detect the expression of HBV DNA Pol. ( C ) TRIM21 and its truncation mutants. ( D ) Huh7 cells were cotransfected with Myc-HBV DNA Pol and FLAG-TRIM21 or its truncation mutants, and co-IP was used to analyze the interaction of Myc-HBV DNA Pol with FLAG-TRIM21 or its truncation mutants. ( E ) GST-HBV DNA Pol and HA-ΔSPRY were transfected into Huh7 cells, and 36 h post transfection, GST beads were used to pull down GST-Pol with its interacting proteins. HBV DNA Pol was detected with anti-GST antibody, and anti-HA antibody was used to detect the expression of ΔSPRY. Data are representative of three independent experiments with three replicates each.

Article Snippet: The full-length sequences of human TRIM21 (Tianjin Saier Biotechnology, SRCL13641) cDNA was obtained by RT-PCR and cloned into the pcDNA3 vector with FLAG, Myc or HA tag between the EcoRI and XhoI sites, and the deletion fragments and point mutants were cloned the same as above.

Techniques: Immunoprecipitation, Expressing, Co-Immunoprecipitation Assay, Transfection

Journal: Viruses

Article Title: The E3 Ubiquitin Ligase TRIM21 Promotes HBV DNA Polymerase Degradation

doi: 10.3390/v12030346

Figure Lengend Snippet: TRIM21 negatively regulates the stability of HBV DNA Pol. ( A ) Huh7 cells were transfected with TRIM21 overexpression or knockdown plasmids or control, and the level of TRIM21 mRNA or protein in the cells was detected by RT-qPCR and Western blot. ( B ) FLAG-HBV DNA Pol was cotransfected with TRIM21 overexpression or knockdown plasmid. The mRNA level of HBV DNA Pol was detected by RT-qPCR. ( C ) Huh7 cells were transfected as described in ( B ), and HepG2.2.15 cells were transfected with TRIM21 overexpression or knockdown plasmids. Western blot was used to detect the protein level of HBV DNA Pol with anti-FLAG antibody or anti-Pol antibody. ( D ) Huh7 cells were transfected with FLAG-HBV DNA Pol along with increasing amounts of TRIM21 expressing plasmid, and WWestern blot was used to detect the protein level of HBV DNA Pol. ( E ) As described in ( C ), except for using pshR-TRIM21 plasmid. ( F ) As described in ( C ), except for using TRIM21-ΔRING plasmid. ( G ) As described in ( C ), except for using TRIM21-ΔSPRY plasmid. ( H ) Huh7 cells were cotransfected with FLAG-HBV DNA Pol and pcDNA3 or TRIM21 plasmid. Thirty hours later, 100μg/ml cycloheximide was used to treat cells, and the protein level of HBV DNA Pol was detected by Western blot. ( I ) Huh7 cells were cotransfected with FLAG-HBV DNA Pol and pSilencer NC or pshR-TRIM21. The treatment was the same as in ( H ). Data are representative of three independent experiments with three replicates each.

Article Snippet: The full-length sequences of human TRIM21 (Tianjin Saier Biotechnology, SRCL13641) cDNA was obtained by RT-PCR and cloned into the pcDNA3 vector with FLAG, Myc or HA tag between the EcoRI and XhoI sites, and the deletion fragments and point mutants were cloned the same as above.

Techniques: Transfection, Over Expression, Knockdown, Control, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Expressing

Journal: Viruses

Article Title: The E3 Ubiquitin Ligase TRIM21 Promotes HBV DNA Polymerase Degradation

doi: 10.3390/v12030346

Figure Lengend Snippet: TRIM21 promotes the degradation of HBV DNA Pol through the ubiquitin proteasome pathway via its RING domain. ( A ) Huh7 cells were transfected with FLAG-HBV DNA Pol expression plasmid. After 30 h, the cells were treated with 20 μM MG132 for 8 h, and Western blot was used to detect the protein level of HBV DNA Pol. ( B ) The cells were transfected as described in ( A ) except for using 0, 25 or 50 μM chloroquine to treat the cells for 6h. ( C ) FLAG-HBV DNA Pol was cotransfected with pcDNA3/pTRIM21 (left panel) or pSilencer-NC/shR-TRIM21 (right panel) in Huh7 cells, and MG132 treatment conditions were the same as in ( A ). Then, Western blot was used to detect the protein level of HBV DNA Pol. ( D ) Huh7 cells were cotransfected with FLAG-HBV DNA Pol and pcDNA3 (left panel)/pSilencer-NC (right panel) and treated with MG132 as described in ( A ), or cotransfected with pTRIM21 (left panel)/shR-TRIM21 (right panel). The protein level of HBV DNA Pol was analyzed by Western blot. ( E ) Huh7 cells were transfected with TRIM21 overexpression plasmid, and MG132 treatment was the same as in ( A ). Then, Western blot was used to detect the protein level of TRIM21. ( F ) The same as in ( B ), except for using chloroquine, and the cells were transfected as described in ( E ). ( G ) Huh7 cells were cotransfected with FLAG-HBV DNA Pol and pcDNA3 or HA-TRIM21, and the cells were treated with 10 μM MG132. Subsequently, anti-FLAG antibody was used to precipitate the proteins for ubiquitination analysis. ( H ) The same as in ( G ), except for pSilencer-NC and shR-TRIM21, which were separately cotransfected with FLAG-HBV DNA Pol. ( I ) Huh7 cells were cotransfected FLAG-HBV DNA Pol with wild-type TRIM21, TRIM21-ΔRING or pcDNA3. The ubiquitin antibody was used to detect the ubiquitination level of HBV DNA Pol, and the anti-FLAG antibody was used to detect the protein level. Data are representative of three independent experiments with three replicates each.

Article Snippet: The full-length sequences of human TRIM21 (Tianjin Saier Biotechnology, SRCL13641) cDNA was obtained by RT-PCR and cloned into the pcDNA3 vector with FLAG, Myc or HA tag between the EcoRI and XhoI sites, and the deletion fragments and point mutants were cloned the same as above.

Techniques: Ubiquitin Proteomics, Transfection, Expressing, Plasmid Preparation, Western Blot, Over Expression

Journal: Viruses

Article Title: The E3 Ubiquitin Ligase TRIM21 Promotes HBV DNA Polymerase Degradation

doi: 10.3390/v12030346

Figure Lengend Snippet: TRIM21 mediates the K48-linked ubiquitination of HBV DNA Pol at K260 and K283. ( A ) A schematic diagram of the lysine point mutations of ubiquitin. ( B ) Overexpression of HBV DNA Pol and TRIM21 in Huh7 cells cotransfected with HA-ubiquitin, Ub-K48R or Ub-K63R mutants. A ubiquitination assay and Western blot were used to detect the ubiquitination and protein level of HBV DNA Pol. ( C ) Schematic of the potential ubiquitination sites of HBV DNA Pol. ( D ) Huh7 cells were transfected with FLAG-HBV DNA Pol or its lysine point mutants, and ubiquitination assays were used to analyze the ubiquitination level of HBV DNA Pol and its lysine mutants. ( E ) TRIM21 and FLAG-HBV DNA Pol or its lysine mutants were cotransfected into Huh7 cells, and the effect of TRIM21 on the ubiquitination level of HBV DNA Pol and its lysine mutants was detected by ubiquitination assay. Data are representative of three independent experiments with three replicates each.

Article Snippet: The full-length sequences of human TRIM21 (Tianjin Saier Biotechnology, SRCL13641) cDNA was obtained by RT-PCR and cloned into the pcDNA3 vector with FLAG, Myc or HA tag between the EcoRI and XhoI sites, and the deletion fragments and point mutants were cloned the same as above.

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

Journal: Viruses

Article Title: The E3 Ubiquitin Ligase TRIM21 Promotes HBV DNA Polymerase Degradation

doi: 10.3390/v12030346

Figure Lengend Snippet: TRIM21 restricts HBV DNA replication. ( A ) Huh7 cells were cotransfected with pHBV1.3 and pcDNA3 or TRIM21 plasmid or pSilencer-NC or shR-TRIM21. qPCR was used to detect HBV DNA. ( B ) HepG2.2.15 cells were transfected with pcDNA3, TRIM21, pSilencer-NC or shR-TRIM21. HBV DNA was detected as described in ( A ). ( C ) HepG2.2.15 cells were transfected with pcDNA3 or TRIM21, and Southern blot was used to detect the cellular replicative intermediate DNA (RI-DNA). ( D ) Huh7 cells were transfected with pHBV1.3, pcDNA3 (left panel) or pTRIM21 (right panel) and ubiquitin or K48R or K63R. HBV DNA was detected by qPCR. ( E ) Except for pHBV1.3, HepG2.2.15 cells were transfected as described in ( D ), and the relative levels of HBV DNA were detected in HepG2.2.15 cells. ( F ) Huh7 cells were cotransfected with pHBV1.3 and pcDNA3 or TRIM21-ΔSPRY, and the HBV DNA level was detected by qPCR. ( G ) Except for pHBV1.3, HepG2.2.15 cells were transfected as described in ( F ). ( H ) HepG2.2.15 cells were transfected with pcDNA3 or TRIM21-ΔSPRY, and RI-DNA was examined by Southern blot. Data are representative of three independent experiments with three replicates each. The data represent the means ± S.D. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The full-length sequences of human TRIM21 (Tianjin Saier Biotechnology, SRCL13641) cDNA was obtained by RT-PCR and cloned into the pcDNA3 vector with FLAG, Myc or HA tag between the EcoRI and XhoI sites, and the deletion fragments and point mutants were cloned the same as above.

Techniques: Plasmid Preparation, Transfection, Southern Blot, Ubiquitin Proteomics

Journal: Viruses

Article Title: The E3 Ubiquitin Ligase TRIM21 Promotes HBV DNA Polymerase Degradation

doi: 10.3390/v12030346

Figure Lengend Snippet: A proposed model describing the role of TRIM21 in the regulation of HBV DNA Pol and HBV DNA replication. HBV-encoded DNA Pol forms a complex with pgRNA and is packaged into a new viral particle by core protein. Some particles reenter into the nucleus and others are enveloped and released from cells for reinfection. Thus, HBV DNA Pol is necessary for HBV replication. Here, we show that TRIM21 mainly promotes the ubiquitination and degradation of HBV DNA Pol, which reduces HBV DNA replication.

Article Snippet: The full-length sequences of human TRIM21 (Tianjin Saier Biotechnology, SRCL13641) cDNA was obtained by RT-PCR and cloned into the pcDNA3 vector with FLAG, Myc or HA tag between the EcoRI and XhoI sites, and the deletion fragments and point mutants were cloned the same as above.

Techniques: 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: Journal of Neuroinflammation

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

doi: 10.1186/1742-2094-11-24

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

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

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

Journal: Journal of Neuroinflammation

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

doi: 10.1186/1742-2094-11-24

Figure Lengend Snippet: List of primers

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

Techniques: Sequencing

Journal: Journal of Neuroinflammation

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

doi: 10.1186/1742-2094-11-24

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

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

Techniques: Infection, SDS Page, Western Blot, Isolation

Journal: Journal of Neuroinflammation

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

doi: 10.1186/1742-2094-11-24

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

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

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

Journal: Journal of Neuroinflammation

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

doi: 10.1186/1742-2094-11-24

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

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

Techniques: Activation Assay, Infection