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