Journal: bioRxiv

Article Title: G 4 C 2 repeat RNA mediates the disassembly of the nuclear pore complex in C9orf72 ALS/FTD

doi: 10.1101/2020.02.13.947721

Figure Lengend Snippet: Western blots and quantification of Trim21 GFP mediated reduction in POM121 ( A-B ), Nup133 ( C-D ), NDC1 ( E-F ), and GAPDH ( G-H ) in wildtype iPSNs. Student’s T-test was used to calculate statistical significance. * p < 0.05, ** p < 0.01.

Article Snippet: On day 18 of differentiation, 5 x 10 6 iPSNs were nucleofected with 5 μg of antibody and 4 μg Trim21 GFP plasmid DNA (Origene) with Lonza P3 Primary Cell 4D Nucleofector Kit (Lonza) using program DC154.

Techniques: Western Blot

Journal: bioRxiv

Article Title: G 4 C 2 repeat RNA mediates the disassembly of the nuclear pore complex in C9orf72 ALS/FTD

doi: 10.1101/2020.02.13.947721

Figure Lengend Snippet: ( A-P ) Maximum intensity projections from SIM imaging ( A ) and quantification ( B-P ) of Nup spots and volume in nuclei isolated from wildtype iPSNs following knockdown of Nup133, POM121, NDC1, or GAPDH. Antibody used for Trim21 mediated knockdown as indicated on left, antibodies as indicated on top. N = 3 wildtype iPSC lines, 50 GFP+ nuclei per line/knockdown. One-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. ** p < 0.01, *** p < 0.001, **** p < 0.0001. ( Q ) Confocal imaging of control iPSNs following Trim21 GFP knockdown of Nup133, POM121, or GAPDH immunostained for Ran. Antibody used for Trim21 knockdown as indicated on left, antibodies as indicated on top. ( R ) Quantification of nuclear to cytoplasmic ratio of Ran. N = 3 wildtype iPSC lines, at least 50 cells per line/knockdown. One-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. **** p < 0.0001. ( S ) Quantification of percent cell death following exposure to glutamate. N = 3 control iPSC lines, 10 frames per well. Two-way ANOVA with Tukey’s multiple comparison test was used to calculate statistical significance. **** p < 0.0001. ( T ) Confocal imaging of cell death in control and C9orf72 iPSNs as measured by propidium iodide (PI) incorporation. Antibody used for Trim Away as indicated on left, glutamate concentration and stain as indicated on top. Scale bar = 5 μm ( A ), 10 μm ( Q ), 100 μm ( T ). * indicates significantly altered Nups.

Article Snippet: On day 18 of differentiation, 5 x 10 6 iPSNs were nucleofected with 5 μg of antibody and 4 μg Trim21 GFP plasmid DNA (Origene) with Lonza P3 Primary Cell 4D Nucleofector Kit (Lonza) using program DC154.

Techniques: Imaging, Isolation, Concentration Assay, Staining

Journal: Life Science Alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: (A) HEK293T cells were transiently transfected with expression constructs for HA-FAT10 and Myc-DDK-TRIM21. After 24 h, cells were harvested and lysed. Cleared lysate was subjected to immunoprecipitation (IP) using FLAG M2 affinity gel, which specifically recognizes the DDK (FLAG) tag. Proteins were visualized by Western blot analysis under reducing conditions (4% 2-ME) using antibodies reactive to HA or FLAG (DDK). GAPDH was used as a loading control. (B) HEK293T cells were transiently co-transfected with expression constructs for HA-FAT10, its conjugation incompetent variant HA–FAT10AV and Myc-DDK-TRIM21. After 24 h, cells were harvested and lysed. Cleared lysate was subjected to immunoprecipitation (IP) using FLAG M2 affinity gel. Proteins were visualized by Western blot analysis under reducing conditions (4% 2-ME) using antibodies reactive to HA or FLAG. GAPDH was used as loading control. (C) In vitro FAT10ylation assay was performed using recombinant proteins. FLAG–UBA6 was incubated with tagless recombinant FAT10 or FAT10AV and C–Myc-DDK-TRIM21 for 30 min at 37°C. Reaction was stopped by adding 4x sample buffer and boiling. SDS–PAGE and subsequent Western blotting was performed using the indicated antibodies under reducing conditions (4% 2-ME). Asterisks indicate TRIM21–FAT10 conjugates. (D) Schematic representation of full-length and truncation mutants of human TRIM21 generated and used in this study. The truncations were constructed using site directed mutagenesis. (E) HEK293T cells were transiently transfected with expression plasmids for HA–FAT10 or HA–FAT10AV and full length or truncation mutants of Myc-DDK-tagged TRIM21. After 24 h, cells were collected and lysed. Cleared cell lysate was subjected to immunoprecipitation using FLAG–M2 affinity gel. SDS–PAGE and Western blot analysis was performed under reducing conditions (4% 2-ME) using antibodies reactive to HA or FLAG (DDK). GAPDH was used as loading control. Shown is one representative experiment out of three independent experiments with similar outcomes for all experiments. Source data are available for this figure.

Article Snippet: Recombinant FLAG-UBA6 was purchased from Enzo Life Sciences and human C-Myc-DDK-TRIM21 was purchased from Origene (#TP302088).

Techniques: Transfection, Expressing, Construct, Immunoprecipitation, FLAG-tag, Western Blot, Control, Conjugation Assay, Variant Assay, In Vitro, Recombinant, Incubation, SDS Page, Generated, Mutagenesis

Journal: Life Science Alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: (A) A549 FLAG-FAT10 cells were infected with IAV (MOI: 1) as indicated and uninfected A549 cells were used as control. After 24 h, cells were harvested and lysed. Precleared cell lysates were subjected to Ni–IDA pull-down assay and incubated overnight at 4°C. Beads were washed four times with lysis buffer. SDS–PAGE under reducing conditions (4% 2-ME) followed by Western blotting was performed using the indicated antibodies. GAPDH was used as the loading control. Asterisk marks nonspecific binding of TRIM21 to the beads. (B) A549 WT and FLAG–FAT10 cells were infected with IAV (MOI: 1), as indicated. After 24 h, cells were harvested and lysed. Cleared cell lysates were subjected to SDS–PAGE under reducing conditions (4% 2-ME) followed by Western blotting using the indicated antibodies. GAPDH was used as the loading control. (C) Densitometric quantification of the fluorescent signal obtained in (B). The total TRIM21 fluorescent signal was normalized to GAPDH. The signal intensity of other samples was calculated relative to uninfected A549 WT cells in which the normalized TRIM21 signal was set to 100%. (D) A549 WT and A549 FLAG-FAT10 cells were infected with IAV (MOI: 1), as indicated. After 24 h, cells were harvested and lysed. Cleared cell lysates were subjected to immunoprecipitation with an antibody reactive to TRIM21. SDS–PAGE under reducing conditions (4% 2-ME) followed by Western blotting was performed using the indicated antibodies. GAPDH was used as loading control. Shown is one representative experiment out of three independent experiments with similar outcomes for each experiment. Error bars in (C) indicate SD (n = 3). * P < 0.05 (one-way anova), ns means not significant. Source data are available for this figure.

Article Snippet: Recombinant FLAG-UBA6 was purchased from Enzo Life Sciences and human C-Myc-DDK-TRIM21 was purchased from Origene (#TP302088).

Techniques: Infection, Control, Pull Down Assay, Incubation, Lysis, SDS Page, Western Blot, Binding Assay, Immunoprecipitation

Journal: Life Science Alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: (A) A549 WT, FLAG–FAT10, TRIM21 KO, TRIM21 KO/FLAG–FAT10 cells were infected with IAV (MOI: 1). After 24 h, supernatants and cell pellets were collected. Cell pellets were lysed and cleared cell lysates were subject to SDS–PAGE under reducing conditions (4% 2-ME) followed by Western blot analysis with the indicated antibodies. GAPDH was used as the loading control. (B) IFNβ ELISA was performed with the cell culture supernatants from (A). (C) A549 WT, FLAG–FAT10, mCherry-TRIM21, mCherry-TRIM21/FLAG–FAT10 cells were infected with IAV (MOI: 1). After 24 h, supernatants and cell pellets were collected. Cell pellets were lysed and cleared cell lysate was subjected to SDS–PAGE under reducing conditions (4% 2-ME) followed by Western blot analysis with the indicated antibodies. GAPDH was used as the loading control. (D) IFNβ ELISA was performed with the cell culture supernatants from (C). (E) A549 WT, TRIM21 KO, mCherry–TRIM21, and FAT10 KO cells were treated with TNF/IFNγ for 24 h followed by IAV infection (MOI: 0.5). After 12 h, supernatant and cell pellets were collected. IFNβ ELISA was performed with the supernatants. Shown is one representative experiment out of at least three independent experiments with similar outcomes for each experiment. Error bars in (B, D, E) indicate SD (n = 3), * P < 0.05 (One-way Anova (B, D), student’s t test (E)), ns means not significant. Source data are available for this figure.

Article Snippet: Recombinant FLAG-UBA6 was purchased from Enzo Life Sciences and human C-Myc-DDK-TRIM21 was purchased from Origene (#TP302088).

Techniques: Infection, SDS Page, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Life Science Alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: (A) HEK293 WT, or where indicated UBA6 KO and USE1 KO cells, were transiently transfected with HA–FAT10 or HA–FAT10AV and Myc–DDK–TRIM21 expression plasmids. After 24 h, cells were collected and lysed. Cleared cell lysate was subjected to SDS–PAGE and subsequent Western blotting under reducing conditions (4% 2-ME) using antibodies reactive to HA or FLAG (DDK). GAPDH was used as loading control. (B) HEK293 WT, or UBA6 KO and USE1 KO cells were transiently transfected with expression constructs for HA–FAT10 and Myc-DDK-TRIM21. Where indicated, HA-UBA6 expression plasmid was transiently transfected in HEK293 UBA6 KO cells, and HIS–USE1 or HIS-USE1 C188A expression plasmids were transiently transfected in HEK293 USE1 KO cells. After 24 h, cells were collected and lysed. Cleared cell lysates were subjected to SDS–PAGE and subsequent Western blotting was performed under reducing conditions (4% 2-ME) using antibodies reactive to HA or FLAG (DDK). GAPDH was used as loading control. In case of UBA6 KO cells, a 4x times amount of the cell lysate was used for immunoprecipitation because overexpression of proteins is always low in this cell line. (C) HEK293 WT, UBA6 KO and USE1 KO cells were transiently transfected with expression constructs for HA–FAT10 and Myc–DDK–TRIM21. Where indicated, cells were treated with 600 U/ml TNF. After 24 h, cells were collected, lysed, and cleared cell lysate was subjected to immunoprecipitation using FLAG M2 affinity gel, which specifically recognizes the DDK (FLAG) tag. SDS–PAGE and Western blotting was performed under reducing conditions (4% 2-ME) using antibodies reactive to HA or FLAG. GAPDH was used as loading control. Shown is one representative experiment out of three independent experiments with similar outcomes for each experiment. Source data are available for this figure.

Article Snippet: Recombinant FLAG-UBA6 was purchased from Enzo Life Sciences and human C-Myc-DDK-TRIM21 was purchased from Origene (#TP302088).

Techniques: Transfection, Expressing, SDS Page, Western Blot, Control, Construct, Plasmid Preparation, Immunoprecipitation, Over Expression, FLAG-tag

Journal: Life Science Alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: (A) HEK293T cells were transiently transfected with expression constructs for HA–FAT10 and Myc–DDK–TRIM21. After 24 h, cells were treated with cycloheximide and/or MG132 for the indicated time points. Cleared cell lysates were subjected to immunoprecipitation using FLAG M2 affinity gel, which is specific for the DDK-tag. SDS–PAGE and Western blot analysis was performed under reducing conditions (4% 2-ME) using antibodies reactive to HA or FLAG (DDK). GAPDH was used as loading control. Shown is one representative experiment out of three independent experiments with similar outcomes. (B) Densitometric quantification of the FAT10-TRIM21 conjugate fluorescent signal, normalized to the respective GAPDH fluorescent signal. The value of untreated sample was set to 100%. Shown is the mean three independent experiments with similar outcomes. Source data are available for this figure.

Article Snippet: Recombinant FLAG-UBA6 was purchased from Enzo Life Sciences and human C-Myc-DDK-TRIM21 was purchased from Origene (#TP302088).

Techniques: Transfection, Expressing, Construct, Immunoprecipitation, SDS Page, Western Blot, Control

Journal: Life Science Alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: (A) A549 cells were treated with TNF/IFNγ for 24 h followed by influenza A virus (IAV) infection (MOI: 1), as indicated. Additional TNF/IFNγ treatment was performed for 24 h. After 24 h, cells were harvested, and cleared cell lysates were subjected to immunoprecipitation with antibodies reactive to TRIM21 or a nonspecific IgG control antibody. Proteins were separated on SDS–PAGE followed by Western blot analysis with the indicated antibodies. GAPDH was used as loading control. Shown is one representative experiment out of three independent experiments with similar outcomes. (B) A549 cells were treated and harvested as indicated in (A). Cleared cell lysates from the lysed cells were immunoprecipitated with antibodies reactive to FAT10 or with a nonspecific IgG control antibody. Proteins were separated on SDS–PAGE followed by Western blot analysis using the indicated antibodies. GAPDH was used as loading control. Shown is one representative experiment out of three independent experiments with similar outcomes. (C) A549 WT and stable FLAG-FAT10 expressing A549 cells were infected with influenza A virus (MOI: 1), as indicated. After 24 h, cells were harvested and lysed. Cleared cell lysates were immunoprecipitated using antibodies reactive to TRIM21 or by using an unspecific IgG control antibody. SDS–PAGE followed by Western blotting was performed using the indicated antibodies. GAPDH was used as loading control. Shown is one representative experiment out of three independent experiments with similar outcomes for each experiment. Source data are available for this figure.

Article Snippet: Recombinant FLAG-UBA6 was purchased from Enzo Life Sciences and human C-Myc-DDK-TRIM21 was purchased from Origene (#TP302088).

Techniques: Virus, Infection, Immunoprecipitation, Control, SDS Page, Western Blot, Expressing

Journal: Life Science Alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: (A) Densitometric quantification of fluorescence signal intensities from . TRIM21 signal intensities were normalized to GAPDH and normalized to the WT + influenza A virus (IAV). Shown is the mean of three independent experiments with similar outcomes. (B) IFNβ ELISA was performed with the cell culture supernatants from uninfected A549 WT, FLAG–FAT10, TRIM21 KO, TRIM21 KO/FLAG–FAT10 cells. (C) A549 WT, FLAG–FAT10, TRIM21 KO, TRIM21 KO/FLAG–FAT10 cells were infected with IAV (MOI: 1). After 24 h, supernatants and cell pellets were collected. Supernatants were used for plaque assay to determine IAV titers. (D) mRNA was isolated from cell pellets obtained in (C) and subjected to real-time PCR analysis. (E) Densitometric quantification of fluorescence signal intensities from . TRIM21 signal intensities were normalized to GAPDH and normalized to the WT + IAV. Shown is the mean of three independent experiments with similar outcomes. (F) IFNβ ELISA was performed with the cell culture supernatants from uninfected A549 WT, FLAG-FAT10, mCherry-TRIM21, mCherry-TRIM21/FLAG–FAT10 cells. (G) A549 WT, FLAG–FAT10, mCherry-TRIM21, mCherry-TRIM21/FLAG–FAT10 cells were infected with IAV (MOI: 1). After 24 h, supernatants and cell pellets were collected. Supernatants were used for plaque assay to determine IAV titers. (H) mRNA was isolated from cell pellets obtained in (E) and subjected to real-time PCR analysis. Error bars in (A, E) indicate SD (n = 3). * P < 0.05 ( t test), ns means not significant. Error bars in (D, H) indicate SD (n = 3). * P < 0.05 (multiple t test).

Article Snippet: Recombinant FLAG-UBA6 was purchased from Enzo Life Sciences and human C-Myc-DDK-TRIM21 was purchased from Origene (#TP302088).

Techniques: Fluorescence, Virus, Enzyme-linked Immunosorbent Assay, Cell Culture, Infection, Plaque Assay, Isolation, Real-time Polymerase Chain Reaction

Journal: Life Science Alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: (A) A549 cells were treated with TNF/IFNγ for 24 h followed by influenza A virus (IAV) infection (MOI: 1), as indicated. Additional TNF/IFNγ treatment was performed for 24 h. Cells were immunostained with antibodies reactive to TRIM21 and FAT10 and visualized by confocal microscopy. Scale bars, 20 μm. (B) Cells from (A) were immunostained with antibodies reactive to M1 and with DAPI. Scale bars, 20 μm. (C) A549 mCherry-TRIM21/FLAG–FAT10 cells were infected with influenza A virus (MOI: 1). Cells were visualized by confocal microscopy. Scale bars, 20 μm. Shown here is one representative experiment out of three independent experiments with similar outcomes.

Article Snippet: Recombinant FLAG-UBA6 was purchased from Enzo Life Sciences and human C-Myc-DDK-TRIM21 was purchased from Origene (#TP302088).

Techniques: Virus, Infection, Confocal Microscopy

Journal: Life Science Alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: (A) Cleared cell lysates from were subjected to SDS–PAGE followed by Western blot analysis using the indicated antibodies. (B) A549 WT, TRIM21 KO, mCherry-TRIM21, and FAT10 KO cells were treated with TNF/IFNγ for 24 h followed by influenza A virus infection (MOI: 0.5). Cells were harvested and cleared cell lysates were subjected to immunoprecipitation using an antibody reactive to FAT10 (4F1, ). SDS–PAGE followed by Western blot analysis was performed using the indicated antibodies. GAPDH was used as the loading control. Shown is one representative experiment out of three independent experiments with similar outcomes. Source data are available for this figure.

Article Snippet: Recombinant FLAG-UBA6 was purchased from Enzo Life Sciences and human C-Myc-DDK-TRIM21 was purchased from Origene (#TP302088).

Techniques: SDS Page, Western Blot, Virus, Infection, Immunoprecipitation, Control

Journal: Life Science Alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: (A) C57BL/6 WT or FAT10KO mice were infected with LCMV (200 pfu), as indicated. Relative FAT10 mRNA expression in mice livers was determined by quantitative real-time PCR. (B) C57BL/6 WT or FAT10KO mice were infected with LCMV (200 pfu), where indicated. Cleared tissue lysates from the livers of the mice were prepared day three post-infection. 40 μg of protein was separated on a SDS–PAGE followed by Western blotting using the indicated antibodies. GAPDH was used as the loading control. (C) Densitometric quantification of fluorescence signal intensities from . TRIM21 signal intensities were normalized to GAPDH. (D) TRIM21 levels in spleen of LCMV infected mice as described in (B). (E) Densitometric quantification of fluorescence signal intensities from . TRIM21 signal intensities were normalized to GAPDH. Shown is one representative experiment out of three independent experiments with similar outcomes. Error bars in (A, C, E) indicate SD (n = 3). * P < 0.05 (one-way Anova). Source data are available for this figure.

Article Snippet: Recombinant FLAG-UBA6 was purchased from Enzo Life Sciences and human C-Myc-DDK-TRIM21 was purchased from Origene (#TP302088).

Techniques: Infection, Expressing, Real-time Polymerase Chain Reaction, SDS Page, Western Blot, Control, Fluorescence

Journal: Life Science Alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: TNF/IFNγ induces the expression of FAT10 (blue arrow). FAT10-mediated down-regulation of type-I IFN happens in two ways: (I) influenza A virus infection upregulates TRIM21 expression (blue arrow). TRIM21 positively regulates the antiviral type-I IFN production through a positive feedback loop (blue arrow). FAT10 inhibits TRIM21 by directly binding to its PRYSPRY domain, causing either degradation of TRIM21 by the 26S proteasome, and/or inhibiting TRIM21 auto-ubiquitination, thus down-regulating the production of type-I IFN. (II) FAT10 gets phosphorylated upon influenza A virus infection. Phosphorylated FAT10 stabilizes and activates OTUB1, which inhibits type-I IFN production .

Article Snippet: Recombinant FLAG-UBA6 was purchased from Enzo Life Sciences and human C-Myc-DDK-TRIM21 was purchased from Origene (#TP302088).

Techniques: Expressing, Virus, Infection, Binding Assay

Journal: Life science alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion.

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: Figure 1. TRIM21 is covalently modified with FAT10. (A) HEK293T cells were transiently transfected with expression constructs for HA-FAT10 and Myc-DDK-TRIM21. After 24 h, cells were harvested and lysed. Cleared lysate was subjected to immunoprecipitation (IP) using FLAG M2 affinity gel, which specifically recognizes the DDK (FLAG) tag. Proteins were visualized by Western blot analysis under reducing conditions (4% 2-ME) using antibodies reactive to HA or FLAG (DDK). GAPDH was used as a loading control. (B) HEK293T cells were transiently co-transfected

Article Snippet: Plasmids used for the transient transfection of HEK293 cells were pCMV6-Myc-DDK-TRIM21 (RC202088; Origene), pcDNA3.1-HA-FAT10 (Hipp et al, 2004), pcDNA3.1-HA-FAT10AV (Aichem et al, 2010), pcDNA3.1-HA-UBA6 (Aichem et al, 2010), pcDNA-3.1-His/-A-USE1, and its active site cysteine mutant pcDNA-3.1-His/-A-USE1-C188A (Aichem et al, 2010), lentiviral envelop plasmid pMD2.G (#12259; Addgene), and lentiviral packaging plasmid psPAX2 (#12260; Addgene). pSMPP-mCherry-hTRIM21 was a gift from Leo James (plasmid #104972 [Clift et al, 2017]; Addgene), and pSpCAS9 wt (BB)2A-GFP targeting human TRIM21 was a gift from Gaudenz Danuser (plasmid # 138295 [Park et al, 2020]; Addgene). pCMV6-Myc-DDKTRIM21 (RC202088; Origene) was used as template to construct the truncation mutations of TRIM21 by site-directed mutagenesis.

Techniques: Transfection, Expressing, Construct, Immunoprecipitation, FLAG-tag, Western Blot, Control

Journal: Life science alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion.

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: Figure 2. FAT10ylation of TRIM21 is catalyzed by the E1 UBA6 and the E2 USE1. (A) HEK293 WT, or where indicated UBA6 KO and USE1 KO cells, were transiently transfected with HA–FAT10 or HA–FAT10AV and Myc–DDK–TRIM21 expression plasmids. After 24 h, cells were collected and lysed. Cleared cell lysate was subjected to SDS–PAGE and subsequent Western blotting under reducing conditions (4% 2-ME) using antibodies reactive to HA or FLAG (DDK). GAPDH was used as loading control. (B) HEK293 WT, or UBA6 KO and USE1 KO cells were transiently transfected with expression

Article Snippet: Plasmids used for the transient transfection of HEK293 cells were pCMV6-Myc-DDK-TRIM21 (RC202088; Origene), pcDNA3.1-HA-FAT10 (Hipp et al, 2004), pcDNA3.1-HA-FAT10AV (Aichem et al, 2010), pcDNA3.1-HA-UBA6 (Aichem et al, 2010), pcDNA-3.1-His/-A-USE1, and its active site cysteine mutant pcDNA-3.1-His/-A-USE1-C188A (Aichem et al, 2010), lentiviral envelop plasmid pMD2.G (#12259; Addgene), and lentiviral packaging plasmid psPAX2 (#12260; Addgene). pSMPP-mCherry-hTRIM21 was a gift from Leo James (plasmid #104972 [Clift et al, 2017]; Addgene), and pSpCAS9 wt (BB)2A-GFP targeting human TRIM21 was a gift from Gaudenz Danuser (plasmid # 138295 [Park et al, 2020]; Addgene). pCMV6-Myc-DDKTRIM21 (RC202088; Origene) was used as template to construct the truncation mutations of TRIM21 by site-directed mutagenesis.

Techniques: Transfection, Expressing, SDS Page, Western Blot, Control

Journal: Life science alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion.

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: Figure 3. FAT10 targets TRIM21 for degradation by the 26S proteasome. (A) HEK293T cells were transiently transfected with expression constructs for HA–FAT10 and Myc–DDK–TRIM21. After 24 h, cells were treated with cycloheximide and/or MG132 for the indicated time points. Cleared cell lysates were subjected to immunoprecipitation using FLAG M2 affinity gel, which is specific for the DDK- tag. SDS–PAGE and Western blot analysis was performed under reducing conditions (4% 2-ME) using antibodies reactive to HA or FLAG (DDK). GAPDH was used as loading control. Shown is one representative experiment out of three independent experiments with similar outcomes. (B) Densitometric quantification of the FAT10-TRIM21 conjugate fluorescent signal, normalized to the respective GAPDH fluorescent signal. The value of untreated sample was set to 100%. Shown is the mean three independent experiments with similar outcomes. Source data are available for this figure.

Article Snippet: Plasmids used for the transient transfection of HEK293 cells were pCMV6-Myc-DDK-TRIM21 (RC202088; Origene), pcDNA3.1-HA-FAT10 (Hipp et al, 2004), pcDNA3.1-HA-FAT10AV (Aichem et al, 2010), pcDNA3.1-HA-UBA6 (Aichem et al, 2010), pcDNA-3.1-His/-A-USE1, and its active site cysteine mutant pcDNA-3.1-His/-A-USE1-C188A (Aichem et al, 2010), lentiviral envelop plasmid pMD2.G (#12259; Addgene), and lentiviral packaging plasmid psPAX2 (#12260; Addgene). pSMPP-mCherry-hTRIM21 was a gift from Leo James (plasmid #104972 [Clift et al, 2017]; Addgene), and pSpCAS9 wt (BB)2A-GFP targeting human TRIM21 was a gift from Gaudenz Danuser (plasmid # 138295 [Park et al, 2020]; Addgene). pCMV6-Myc-DDKTRIM21 (RC202088; Origene) was used as template to construct the truncation mutations of TRIM21 by site-directed mutagenesis.

Techniques: Transfection, Expressing, Construct, Immunoprecipitation, SDS Page, Western Blot, Control

Journal: Life science alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion.

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: Figure 4. FAT10 modulates the stability of TRIM21 upon influenza A virus (IAV) infection. (A) A549 FLAG-FAT10 cells were infected with IAV (MOI: 1) as indicated and uninfected A549 cells were used as control. After 24 h, cells were harvested and lysed. Precleared cell lysates were subjected to Ni–IDA pull-down assay and incubated overnight at 4°C. Beads were washed four times with lysis buffer. SDS–PAGE under reducing conditions (4% 2-ME) followed by Western blotting was performed using the indicated antibodies. GAPDH was used as the loading control. Asterisk marks

Article Snippet: Plasmids used for the transient transfection of HEK293 cells were pCMV6-Myc-DDK-TRIM21 (RC202088; Origene), pcDNA3.1-HA-FAT10 (Hipp et al, 2004), pcDNA3.1-HA-FAT10AV (Aichem et al, 2010), pcDNA3.1-HA-UBA6 (Aichem et al, 2010), pcDNA-3.1-His/-A-USE1, and its active site cysteine mutant pcDNA-3.1-His/-A-USE1-C188A (Aichem et al, 2010), lentiviral envelop plasmid pMD2.G (#12259; Addgene), and lentiviral packaging plasmid psPAX2 (#12260; Addgene). pSMPP-mCherry-hTRIM21 was a gift from Leo James (plasmid #104972 [Clift et al, 2017]; Addgene), and pSpCAS9 wt (BB)2A-GFP targeting human TRIM21 was a gift from Gaudenz Danuser (plasmid # 138295 [Park et al, 2020]; Addgene). pCMV6-Myc-DDKTRIM21 (RC202088; Origene) was used as template to construct the truncation mutations of TRIM21 by site-directed mutagenesis.

Techniques: Virus, Infection, Control, Pull Down Assay, Incubation, Lysis, SDS Page, Western Blot

Journal: Life science alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion.

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: Figure 5. FAT10-mediated inhibition of TRIM21 down-regulates IFNβ secretion upon influenza A virus (IAV) infection. (A) A549 WT, FLAG–FAT10, TRIM21 KO, TRIM21 KO/FLAG–FAT10 cells were infected with IAV (MOI: 1). After 24 h, supernatants and cell pellets were collected. Cell pellets were lysed and cleared cell lysates were subject to SDS–PAGE under reducing conditions (4% 2-ME) followed by Western blot analysis with the indicated antibodies. GAPDH was used as the loading control. (B) IFNβ ELISA was performed with the cell culture supernatants from (A). (C) A549 WT, FLAG–FAT10, mCherry-TRIM21, mCherry-

Article Snippet: Plasmids used for the transient transfection of HEK293 cells were pCMV6-Myc-DDK-TRIM21 (RC202088; Origene), pcDNA3.1-HA-FAT10 (Hipp et al, 2004), pcDNA3.1-HA-FAT10AV (Aichem et al, 2010), pcDNA3.1-HA-UBA6 (Aichem et al, 2010), pcDNA-3.1-His/-A-USE1, and its active site cysteine mutant pcDNA-3.1-His/-A-USE1-C188A (Aichem et al, 2010), lentiviral envelop plasmid pMD2.G (#12259; Addgene), and lentiviral packaging plasmid psPAX2 (#12260; Addgene). pSMPP-mCherry-hTRIM21 was a gift from Leo James (plasmid #104972 [Clift et al, 2017]; Addgene), and pSpCAS9 wt (BB)2A-GFP targeting human TRIM21 was a gift from Gaudenz Danuser (plasmid # 138295 [Park et al, 2020]; Addgene). pCMV6-Myc-DDKTRIM21 (RC202088; Origene) was used as template to construct the truncation mutations of TRIM21 by site-directed mutagenesis.

Techniques: Inhibition, Virus, Infection, SDS Page, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Life science alliance

Article Title: FAT10 inhibits TRIM21 to down-regulate antiviral type-I interferon secretion.

doi: 10.26508/lsa.202402786

Figure Lengend Snippet: Figure 6. Cartoon summarizing FAT10 mediated inhibition of type-I IFN signaling. TNF/IFNγ induces the expression of FAT10 (blue arrow). FAT10-mediated down-regulation of type-I IFN happens in two ways: (I) influenza A virus infection upregulates TRIM21 expression (blue arrow). TRIM21 positively regulates the antiviral type-I IFN production through a positive feedback loop (blue arrow). FAT10 inhibits TRIM21 by directly binding to its PRYSPRY domain, causing either degradation of TRIM21 by the 26S proteasome, and/or inhibiting TRIM21 auto- ubiquitination, thus down-regulating the production of type-I IFN. (II) FAT10 gets phosphorylated upon influenza A virus infection. Phosphorylated FAT10 stabilizes and activates OTUB1, which inhibits type-I IFN production (Saxena et al, 2024).

Article Snippet: Plasmids used for the transient transfection of HEK293 cells were pCMV6-Myc-DDK-TRIM21 (RC202088; Origene), pcDNA3.1-HA-FAT10 (Hipp et al, 2004), pcDNA3.1-HA-FAT10AV (Aichem et al, 2010), pcDNA3.1-HA-UBA6 (Aichem et al, 2010), pcDNA-3.1-His/-A-USE1, and its active site cysteine mutant pcDNA-3.1-His/-A-USE1-C188A (Aichem et al, 2010), lentiviral envelop plasmid pMD2.G (#12259; Addgene), and lentiviral packaging plasmid psPAX2 (#12260; Addgene). pSMPP-mCherry-hTRIM21 was a gift from Leo James (plasmid #104972 [Clift et al, 2017]; Addgene), and pSpCAS9 wt (BB)2A-GFP targeting human TRIM21 was a gift from Gaudenz Danuser (plasmid # 138295 [Park et al, 2020]; Addgene). pCMV6-Myc-DDKTRIM21 (RC202088; Origene) was used as template to construct the truncation mutations of TRIM21 by site-directed mutagenesis.

Techniques: Inhibition, Expressing, Virus, Infection, Binding Assay, Ubiquitin Proteomics

Journal: Advanced Science

Article Title: Anesthetic Propofol Promotes Tumor Metastasis in Lungs via GABA A R‐Dependent TRIM21 Modulation of Src Expression

doi: 10.1002/advs.202102079

Figure Lengend Snippet: TRIM21 regulates Src protein level to mediate GABA A R signaling. Western blot analysis of TRIM21 expression in HCT116 cells treated with a) propofol, b) muscimol, or d) bicuculine. c) qRT‐PCR analysis of TRIM21 mRNA expression in HCT116 cells treated with propofol or control condition ( N = 3, mean ± SD, Student's t ‐test, P > 0.5). e) Western blot analysis of Src and p‐Src expression in HCT116 cells with or without overexpression of TRIM21. GAPDH is a loading control. f) qRT‐PCR analysis of Src mRNA expression in HCT116 cells with or without overexpression of TRIM21 ( N = 3, mean ± SD, Student's t ‐test, P > 0.5). g) Fluorescent micrographs of adhesion between the HUVEC monolayer and the HCT116 cells (green) with or without overexpression of TRIM21 and Src (scale bar = 100 µm). h) Quantification of the HCT116 cells adhesion to the HUVECs monolayer ( N = 6, mean ± SD, one‐way ANOVA test with post‐hoc Bonferroni test, F = 11.04, P = 0.001). i) F‐actin labeling of membrane protrusions in HCT116 cells with or without overexpression of TRIM21 and Src (scale bar = 100 µm). Arrows indicate extensions of cell membrane protrusions. j) Immunofluorescence staining of Talin and Vinculin in HCT116 cells with or without overexpression of TRIM21 and Src (scale bar = 50 µm). Arrows indicate focal adhesions. k) Quantification of the membrane‐localized Talin and Vinculin in focal adhesion ( N = 4, mean ± SD, one‐way ANOVA test with post‐hoc Bonferroni test, F = 25.24, P < 0.001). l) Western blot analysis of Src and phosphorylated Src (p‐Src) expression in HCT116 cells with or without overexpression of TRIM21 and following propofol or control condition. GAPDH is a loading control. m) Fluorescent micrographs of the adhesion between the HUVEC monolayer and the HCT116 cells (green) with or without overexpression of TRIM21 and treated with propofol or control condition (scale bar = 100 µm). n) Fluorescent micrographs showing adhesion of muscimol‐treated HCT116 cells (green) with or without TRIM21 overexpression to the HUVEC monolayer (scale bar = 100 µm). o) Quantification of panel (n) ( N = 6, mean ± SD, Student's t ‐test, P = 0.002). p) F‐actin labeling of membrane protrusions in muscimol‐treated HCT116 cells with or without TRIM21 overexpression (scale bar = 100 µm). Arrows indicate extensions of cell membrane protrusions. q) Immunofluorescence staining of Talin and Vinculin in muscimol‐treated HCT116 cells with or without TRIM21 overexpression (scale bar = 50 µm). Arrows indicate focal adhesions. Ctrl, control. * P < 0.05, *** P < 0.001, # P < 0.05, and ## P < 0.01. Ctrl, control; HUVEC, human umbilical vein endothelial cell; SD, standard deviation; p‐Src, phosphorylated Src.

Article Snippet: Virus was packaged by 293FT cells and used to infect HCT116 cells. pCMV3‐TRIM21 with C‐HA (HG18010‐CY, Sino Biological, Beijing, China) or pCMV3‐TRIM21 with C‐Myc (HG18010‐CM, Sino Biological) was used for human TRIM21 overexpression. pCMV3‐Src with C‐Flag (HG29841‐CF, Sino Biological) was used for human Src overexpression.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Over Expression, Labeling, Immunofluorescence, Staining, Standard Deviation

Journal: Advanced Science

Article Title: Anesthetic Propofol Promotes Tumor Metastasis in Lungs via GABA A R‐Dependent TRIM21 Modulation of Src Expression

doi: 10.1002/advs.202102079

Figure Lengend Snippet: The hypothesized pathway indicating propofol promotes tumor metastasis. The hypothesized pathway indicating that propofol activated GABA A R to downregulate TRIM21 and consequently upregulate Src, leading to enhancement of adhesion and extension of the tumor cells with VECs and promotion of tumor metastasis in lungs of mice. Schematic created at BioRender.com with permission.

Article Snippet: Virus was packaged by 293FT cells and used to infect HCT116 cells. pCMV3‐TRIM21 with C‐HA (HG18010‐CY, Sino Biological, Beijing, China) or pCMV3‐TRIM21 with C‐Myc (HG18010‐CM, Sino Biological) was used for human TRIM21 overexpression. pCMV3‐Src with C‐Flag (HG29841‐CF, Sino Biological) was used for human Src overexpression.

Techniques:

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