Journal: Theranostics

Article Title: E3 ubiquitin ligase TRIM21 targets TIF1γ to regulate β-catenin signaling in glioblastoma

doi: 10.7150/thno.85662

Figure Lengend Snippet: TRIM21 is highly expressed in GBM samples and correlates with poor prognosis. (A) Venn diagram showing the number of changed proteins from TRIMs family identified in TCGA-datasets in GBM relative to normal brain tissue samples (Blue) and the prognosis difference using Kaplan-Meier curves in GBM from TCGA-database (Yellow) and Rembrandt-Database (Green) and overlapping proteins in the datasets. (B) TRIM21 and TRIM48 were identified. (C) Western blotting of TRIM21 and TRIM48 protein in 5 paired surgically removed glioma (T) and the corresponding adjacent normal tissues (N). (D) The mRNA of TRIM21 in glioma in different grades. (E-F) Representative IHC staining images (E) and score (F) of TRIM21 in WHO grade I-IV glioma tissue microarrays and normal brain tissues (NBT). Scale bar, 100 μm. (G-H) Kaplan-Meier curves showing the relationship between the expression of TRIM21 and the overall survival rate (G) and Disease-free survival rate (H) of patients with glioma (n = 120). (I) Heatmap showing the distribution of clinical features and genetic characteristics and TRIM21 expressions in glioma specimens from TCGA-LGGGBM database (n = 330).

Article Snippet: His-tagged β-catenin and/or His-tagged TRIM21 (MCE HY-P71791) were added to the immobilized GST-His-TIF1γ and incubated overnight at 4 °C under gentle rotation.

Techniques: Western Blot, Immunohistochemistry, Expressing

Journal: Theranostics

Article Title: E3 ubiquitin ligase TRIM21 targets TIF1γ to regulate β-catenin signaling in glioblastoma

doi: 10.7150/thno.85662

Figure Lengend Snippet: The correlation between TRIM21 expression and clinicopathological factors of patients with glioma.

Article Snippet: His-tagged β-catenin and/or His-tagged TRIM21 (MCE HY-P71791) were added to the immobilized GST-His-TIF1γ and incubated overnight at 4 °C under gentle rotation.

Techniques: Expressing

Journal: Theranostics

Article Title: E3 ubiquitin ligase TRIM21 targets TIF1γ to regulate β-catenin signaling in glioblastoma

doi: 10.7150/thno.85662

Figure Lengend Snippet: TRIM21 facilitated invasion and growth of GBM via its RING domain in vivo and in vitro . (A) Expression of TRIM21 protein in GBM cells (GBM-1 and U87-MG) transfected with Scramble or sh TRIM21 . (B) The growth curve of GBM cells GBM cells (GBM-1 and U87-MG) transfected with Scramble or sh TRIM21 . (C) Representative images ( Left panel ) and number ( Right panel ) of U87-MG cells transfected with Scramble or sh TRIM21 . Scar bars, 50μm. (D) Western blot analysis of TRIM21 in GBM cells (U87-MG and U251-MG) transfected with Mock, TRIM21-FL, TRIM21-△RING plasmid. (E) Representative images ( Left panel ) and number ( Right panel ) of Mock, TRIM21-FL, TRIM21-△RING GBM cells (U87-MG and U251-MG) in transwell assay. (F-G) Summary graph indicates the motilities (F) and Brdu-positive immunofluorescence staining (G) of GBM cells transfected with Mock, TRIM21-FL, TRIM21-△RING plasmid. (H-I) Bioluminescent images (H) and the quantification ( I ) of tumors in mice implanted with Mock-, TRIM21-FL-, or TRIM21-△RING- U87-MG cells. The bioluminescent images were obtained at day 7 and 14 after injection. (J) Survival curves of tumor-bearing mice implanted with indicated cells. (K) Representative IHC staining of the tumor tissues were performed with anti-Ki-67 antibodies. Scar bars, 100μm

Article Snippet: His-tagged β-catenin and/or His-tagged TRIM21 (MCE HY-P71791) were added to the immobilized GST-His-TIF1γ and incubated overnight at 4 °C under gentle rotation.

Techniques: In Vivo, In Vitro, Expressing, Transfection, Western Blot, Plasmid Preparation, Transwell Assay, Immunofluorescence, Staining, Injection, Immunohistochemistry

Journal: Theranostics

Article Title: E3 ubiquitin ligase TRIM21 targets TIF1γ to regulate β-catenin signaling in glioblastoma

doi: 10.7150/thno.85662

Figure Lengend Snippet: TRIM21-mediated WNT/β-catenin through K63 polyubiquitination of β-catenin. (A) GSEA of positive regulation of WNT signaling pathway score in human GBMs from the GSE108474 (n = 110). (B) Gene oncology enrichment analysis of identified Differential Expression Analysis (DEG) by TMT from U87-MG. (C) Western blot analysis to evaluate β-catenin and C-myc in lysates prepared from mock, TRIM21-FL and TRIM21-△RING-GBM (U87-MG and U251-MG) cells. (D) Western blot analysis to evaluate β-catenin and C-myc in lysates prepared from TRIM21 knock down. (E) Immunofluorescence ( left ) and quantitative analysis ( right ) of β-catenin nuclear translocation induced by overexpression of TRIM21. Scale bar,20 µm. (F-G) Overexpression (F) and silencing (G) of TRIM21 enhanced and abolished the nuclear translocation of β-catenin, respectively. β-catenin protein in the cytoplasm and nucleus were assayed in U87-MG cells with up- or down- of TRIM21 by a nuclear extraction assay. (H) Immunoprecipitation was performed with U87-MG-OE TRIM21 -Flag ( Left panel ) and HEK293-T ( Right panel ) lysates using Flag or β-catenin antibodies, and the precipitants were measured by western blotting with the indicated antibodies. (I) The Armadillo domain of β-catenin mediates the interaction of this protein with TRIM21 ( Top panel ), Schematic illustration of β-catenin. Bottom panel, β-Catenin deletion mutants were co-expressed with HA-TRIM21 in 293T cells. the cells were subjected to IP with a HA antibody followed by immunoblotting (IB) with FLAG and HA antibodies. (J) In vivo ubiquitination assays performed in HEK 293T cells with mock, TRIM21, TRIM21-△RING plasmid, TRIM21-C16A, which then were transfected as indicated. Cells were treated with MG132 (10 mM for 4 h) and prior to lysis and then subjected to anti-β-catenin IP followed by anti-UB with immunoblot analysis. (K) His- Ub (WT, K6R, K27R, K29R, K33R, K48R, K63R) mutants were expressed with or without HA-TRIM21 in HEK293T cells. (L) HEK293T cells were transfected with Flag-β-catenin and HA-TRIM21 together with wild-type (WT) ubiquitin with K48O and K63O respectively. Cells were treated with MG132 (10 mM for 4 h) and prior to lysis and then subjected to anti-β-catenin IP followed by anti-His with immunoblot analysis.

Article Snippet: His-tagged β-catenin and/or His-tagged TRIM21 (MCE HY-P71791) were added to the immobilized GST-His-TIF1γ and incubated overnight at 4 °C under gentle rotation.

Techniques: Quantitative Proteomics, Western Blot, Knockdown, Immunofluorescence, Translocation Assay, Over Expression, Extraction, Immunoprecipitation, In Vivo, Ubiquitin Proteomics, Plasmid Preparation, Transfection, Lysis

Journal: Theranostics

Article Title: E3 ubiquitin ligase TRIM21 targets TIF1γ to regulate β-catenin signaling in glioblastoma

doi: 10.7150/thno.85662

Figure Lengend Snippet: TRIM21-mediated degradation of TIF1γ. (A) Volcano plot of the protein abundance changes in response to OE TRIM21 . Average protein expression ratio of 4 replicates (log 2 transformed) between TRIM21 overexpression and mock U87-MG cells was plotted against p-value by t-test (-log 10 transformed). Cutoffs of p = 0.05 and 2.0 -fold change were marked, respectively. The figure below shows the total number of proteins identified as well as the number of up- and down-regulated proteins. (B) MS analysis identified TIF1γ as the key interacting protein of TRIM21. TRIM21 was immunoprecipitated from U87-MG expressing HA-TRIM21 using anti-HA-conjugated beads. TIF1γ was identified through MS analysis by peptides covering the TIF1γ protein sequence. representative detected peptide (QIDLVDNYFVK) of TIF1γ was shown. (C) Co-IP of TRIM21 with TIF1γ in U87 cells ( left panel ) and U251-cells ( Right panel ). (D) Co-IP of TIF1γ with TRIM21 in U87 cells ( left panel ) and U251-cells ( Right panel ). (E) Full-length TRIM21 and a series of TRIM21 mutants with deletion (△) of various domains ( top panel ). 293 T cells were co-transfected with Flag-TIF1γ and WT HA-TRIM21 or their truncation mutants for 48 h. CO-IP assay was performed. (F) Full-length TIF1γ and a series of TIF1γ mutants with deletion (△) of various domains ( top panel ). 293 T cells were co-transfected with Flag-TIF1γ or their truncation mutants and WT HA-TRIM21 CO-IP assay was performed. (G) In vivo ubiquitination assays performed in U87-Flag- TIF1γ cells transfected with indicated plasmid. Cells were treated with MG132 (10 mM for 4 h) and prior to lysis and then subjected to anti-Flag IP followed by anti-UB with immunoblot analysis. (H-I) TRIM21 is a K48-linkage-specificubiquitin ligase for TIF1γ. HEK293T cells were transfected with Flag-TIF1γ and HA-TRIM21 together with wild-type (WT) ubiquitin (H) , with each of the ubiquitin mutants Ub K48O , Ub K63O (H), Ub K48R or Ub K63R (I) . (J) HEK293-T cells were cotransfected with the indicated plasmids for 48 h and then treated with 10 mM MG132. The polyubiquitination levels of Flag-TIF1γ and Flag-TIF1γ-K5R; Flag-TIF1γ-K1115R mutant protein were analyzed. (K) Left panel: U87-MG cells were transfected with TRIM21-TIF1γ-WT or TRIM21-TIF1γ-K5R plasmid for 24 h, and then treated with cycloheximide (CHX, 20 μg/mL) or the indicated times before harvesting. Cells lysates were analyzed by immunoblotting.

Article Snippet: His-tagged β-catenin and/or His-tagged TRIM21 (MCE HY-P71791) were added to the immobilized GST-His-TIF1γ and incubated overnight at 4 °C under gentle rotation.

Techniques: Quantitative Proteomics, Expressing, Transformation Assay, Over Expression, Immunoprecipitation, Sequencing, Co-Immunoprecipitation Assay, Transfection, In Vivo, Ubiquitin Proteomics, Plasmid Preparation, Lysis, Western Blot, Mutagenesis

Journal: Theranostics

Article Title: E3 ubiquitin ligase TRIM21 targets TIF1γ to regulate β-catenin signaling in glioblastoma

doi: 10.7150/thno.85662

Figure Lengend Snippet: TRIM21 / TIF1γ axis is involved in β-catenin stability and tumor progression. (A) Immunoblotting of TRIM21, TIF1γ and β-catenin in GBM cells (U87-MG and U251-MG) overexpressing TRIM21, TIF1γ and TRIM21 + TIF1γ. (B) Immunofluorescence scanning of β-catenin (Red), TIF1γ(Green), TRIM21(White) in GBM cells overexpressing TRIM21 and/or TIF1γ; Scar bars, 50μm. (C) In vivo ubiquitination assays performed in U87-MG cells transfected with indicated plasmid. Cells were treated with MG132 (10 mM for 4 h) and prior to lysis and then subjected to anti-β-catenin IP followed by anti-UB with immunoblot analysis. (D) Immunoblotting of TRIM21, TIF1γ in U87-MG-siTIF1γ cells Overexpressing TIF1γ, TI F1 γ-K5R, TRIM21+ TIF1γ, TIF1γ-K5R+ TRIM21. (E) The invasive number of Mock, OE TRIM21 , OE TIF1γ and OE TRIM21+TIF1γ GBM Cells (U87-MG and U251-MG). (F) The number of Brdu-positive immunofluorescence staining for the indicated GBM cells. (G-H) Bioluminescence (G) and Quantification (H) of tumors in NOD-SCID mice implanted with Mock, TRIM21, TIF1γ and TIF1γ+ TRIM21-U87-MG cells. (I) Survival curves of tumor-bearing mice implanted with Mock, OE TRIM21 , OE TIF1γ and OE TIF1γ+TRIM21 U87-MG cells. (J) Representative IHC image of the tumor tissues were performed with anti-β-catenin and anti-Ki-67 antibodies. Scar bars, 50μm.

Article Snippet: His-tagged β-catenin and/or His-tagged TRIM21 (MCE HY-P71791) were added to the immobilized GST-His-TIF1γ and incubated overnight at 4 °C under gentle rotation.

Techniques: Western Blot, Immunofluorescence, In Vivo, Ubiquitin Proteomics, Transfection, Plasmid Preparation, Lysis, Staining

Journal: Theranostics

Article Title: E3 ubiquitin ligase TRIM21 targets TIF1γ to regulate β-catenin signaling in glioblastoma

doi: 10.7150/thno.85662

Figure Lengend Snippet: Arginine 443 in TRIM21 is critical for mediating the ubiquitination and degradation of TIF1γ. (A) The R443W mutation of TRIM21 was identified from TCGA GBM database ( http://www.cbioportal.org ). (B) U87-MG cells were transfected with HA-tagged TRIM21 and its mutants R443W (HA-TRIM21, HA-TRIM21 R443W respectively) Cell lysates were immunoprecipitated with anti-HA antibodies. (C) U87-MG cells were transfected with HA-tagged TRIM21, its mutants HA-TRIM21-R443W and/or Flag-TIF1γ respectively, cells were immunoprecipitated with anti-TIF1γ. (D) Western blot analysis to evaluate TIF1γ, β-catenin, C-myc and cyclin D1 in lysates prepared from Mock, TRIM21 and TRIM21-R443W-GBM (U87-MG) cells. (E) In vivo ubiquitination assays performed in U87-MG transfected with mock; HA-tagged TRIM21, its mutants HA-TRIM21-R443W. Cells were treated with MG132 (10 mM for 4 h) and prior to lysis and then subjected to anti-Flag IP followed by anti-HIS with immunoblot analysis. (F) Immunoblotting ( Left panel ) and Quantitation ( Right panel ) of TIF1γ in GBM cell (Mock, TRIM21, TRIM21 R443W ) treated with CHX (100 mg/ml) and MG132 for different times. (G) Brdu-positive immunofluorescence staining images ( Upper panel ) and number ( down panel ) of GBM cells transfected with (-Mock, -TRIM21, -TRIM21 R443W) Scar bars, 100μm. (H) Representative images ( Upper panel ) and number ( down panel ) of invaded U87-MG cells and U251-MG transfected with (-Mock, -TRIM21, -TRIM21 R443W ) Scar bars, 100μm.

Article Snippet: His-tagged β-catenin and/or His-tagged TRIM21 (MCE HY-P71791) were added to the immobilized GST-His-TIF1γ and incubated overnight at 4 °C under gentle rotation.

Techniques: Ubiquitin Proteomics, Mutagenesis, Transfection, Immunoprecipitation, Western Blot, In Vivo, Lysis, Quantitation Assay, Immunofluorescence, Staining

Journal: Theranostics

Article Title: E3 ubiquitin ligase TRIM21 targets TIF1γ to regulate β-catenin signaling in glioblastoma

doi: 10.7150/thno.85662

Figure Lengend Snippet: Correlation of TRIM21, TIF1γ and β-catenin in Glioma. (A-D) Representative image of TRIM21, TIF1γ and β-catenin expression in IDH1-WT and IDH1-MT glioma examined by IHC. Scare bars, 100 μm. (E-F) Kaplan-Meier plots of the OS (E) rates and DFS (F) rates in human glioma specimens in the groups with high and low expression of TIF1γ. (G) IHC staining of 120 human glioma specimens was performed with the indicated antibodies. Representative images from the staining of TRIM21, TIF1γ and β-catenin were shown scar bars, 100μm. (H) χ 2 analysis showing the percentage of TIF1γ-high and TIF1γ-low in TRIM21-high group and TRIM21-low group. (I) χ2 analysis showing the percentage of β-catenin-high and β-catenin-low in TRIM21-high group and TRIM21-low group. (J) χ2 analysis showing the percentage of β-catenin-high and β-catenin-low in TIF1γ-high group and TIF1γ-low group. (K) Kaplan-Meier plots of the overall survival rates and Disease-free survival rates in human glioma specimens in the groups with TRIM21(+) β‐catenin (+), TRIM21(-) β‐catenin (-), and others.

Article Snippet: His-tagged β-catenin and/or His-tagged TRIM21 (MCE HY-P71791) were added to the immobilized GST-His-TIF1γ and incubated overnight at 4 °C under gentle rotation.

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Theranostics

Article Title: E3 ubiquitin ligase TRIM21 targets TIF1γ to regulate β-catenin signaling in glioblastoma

doi: 10.7150/thno.85662

Figure Lengend Snippet: Inhibition of xenograft tumor proliferation by in vivo treatment with siRNA. (A-C) U87-MG cells (1 × 10 6 cells) were subcutaneously implanted into nude mice. After 10 days, control or TRIM21 siRNA was injected into the developed xenograft tumors (n = 5). 10 days after siRNA injection, the nude mice were killed. Dashed lines show the outline of xenograft tumors in representative three mice ( Left picture ), and extirpated xenograft tumors are shown ( Right pictur e) (A) . The volumes (B) and weights (C) of five pair of tumors were measured. (D) Sections were stained with TRIM21 Scar bars, 50 μm.

Article Snippet: His-tagged β-catenin and/or His-tagged TRIM21 (MCE HY-P71791) were added to the immobilized GST-His-TIF1γ and incubated overnight at 4 °C under gentle rotation.

Techniques: Inhibition, In Vivo, Control, Injection, Staining

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: 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: STAR Protocols

Article Title: In vivo optical recordings of ion dynamics in mouse corneal primary nociceptive terminals

doi: 10.1016/j.xpro.2022.101224

Figure Lengend Snippet:

Article Snippet: AAV1.Syn.TurboRFP.WPRE.RBG , James M. Wilson , Addgene AAV1; 105552-AAV1; RRID: Addgene_105552 http://addgene.org/105552.

Techniques: Recombinant, Software, Imaging, Fluorescence, In Vivo, Microscopy, Transferring