Journal: The Journal of Biological Chemistry

Article Title: Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4 CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration *

doi: 10.1074/jbc.M116.733626

Figure Lengend Snippet: ACR and the AL2CR bind an active CRL4CRBN. A, St, St-ACR, St-AL1CR, and St-AL2CR mouse brain pulldowns were incubated with (+) or without (−) ubiquitin-FLAG (Ub-FLAG), E2, and/or E1. After the reaction, these in vitro ubiquitination assays were probed on WB with an anti-FLAG antibody. E2 promotes ubiquitination in St-ACR and St-AL2CR pulldowns. The WB shown is representative of five independent experiments. B, WB with an α-CRBN antibody indicates the in vitro ubiquitination assays of St-ACR brain pulldowns where Crbn is poly-ubiquitinated, indicating that St-ACR isolates from brain an active CRL4CRBN. The WB shown is representative of two independent experiments.

Article Snippet: In Vitro Ubiquitination Assay Pulldown samples were incubated in 50 m m Tris, pH 7.6, 5 m m MgCl 2 , 2 m m ATP, 0.6 m m DTT, with/without 40 ng of the E1 UBE1 (catalog no. E-305, lot 16114714, BostonBiochem, Cambridge, MA), 0.3 μg of the E2 UbcH5a/UBE2D1 (catalog no. E2-616, Lot 04201314C, BostonBiochem, Cambridge, MA), 1 μg of ubiquitin-FLAG (catalog no. U-211, Lot DBGI0215011, BostonBiochem), 1 μ m recombinant human HA ubiquitin aldehyde, C terminus (catalog no. U-556, lot 0AB03101C, BostonBiochem).

Techniques: Incubation, In Vitro

Journal: The Journal of Biological Chemistry

Article Title: Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4 CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration *

doi: 10.1074/jbc.M116.733626

Figure Lengend Snippet: In vitro ubiquitination of proteins present in the St-ACR Tyr(P)Thr(P) pulldowns 1st column lists some of the proteins ubiquitinated in vitro in an ACR Tyr(P)Thr(P) -dependent manner. 2nd column lists all the lysine residues found ubiquitinated in vitro. The K-ub found in our UbiScans from mouse brains are indicated with (m*) and in previous UbiScan experiments from mouse (m), human (h), rat (r) and chicken (c) tissues, which are reported online. 3rd column reports database accession numbers.

Article Snippet: In Vitro Ubiquitination Assay Pulldown samples were incubated in 50 m m Tris, pH 7.6, 5 m m MgCl 2 , 2 m m ATP, 0.6 m m DTT, with/without 40 ng of the E1 UBE1 (catalog no. E-305, lot 16114714, BostonBiochem, Cambridge, MA), 0.3 μg of the E2 UbcH5a/UBE2D1 (catalog no. E2-616, Lot 04201314C, BostonBiochem, Cambridge, MA), 1 μg of ubiquitin-FLAG (catalog no. U-211, Lot DBGI0215011, BostonBiochem), 1 μ m recombinant human HA ubiquitin aldehyde, C terminus (catalog no. U-556, lot 0AB03101C, BostonBiochem).

Techniques: In Vitro

Journal: The Journal of Biological Chemistry

Article Title: Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4 CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration *

doi: 10.1074/jbc.M116.733626

Figure Lengend Snippet: APP could be both a substrate and a substrate recognition subunit of Stub1 and CRL4CRBN E3 protein ligases. A, CRL4CRBN mediates ubiquitination of lysine residue(s) present in the cytoplasmic tail of APP (with Lys756 being the most likely candidate). B, Stub1 could also be involved in the ubiquitination of cytoplasmic APP lysine residue(s). C, APP could bridge cytosolic and membrane-bound proteins to CRL4CRBN de facto functioning as a substrate recognition unit of a CRL4CRBN-APP E3 ubiquitin-protein ligase. D, in this final model APP is postulated to act as a substrate recognition unit for a Stub1-APP E3 ubiquitin-protein ligase, mediating ubiquitination of cytosolic and/or membrane-bound proteins that interact with the ACR ubiquitination of APP, and APP-binding proteins could lead to functional modification and/or to degradation of the ubiquitinated proteins by the proteasome, autophagosomes, and/or lysosomes. E, some of the APP-derived metabolites that contain the ACR and can potentially interact with Stub1 and CRL4CRBN. Processing of full-length APP by β-, α-, and γ-secretase can have several functional consequences. For example, it is possible that full-length APP-Stub1/CRL4CRBN, β-CTF-Stub1/CRL4CRBN, and α-CTF-Stub1/CRL4CRBN complexes have distinct functions, i.e. that the ectodomain of APP may influence the function of the ACR. That processing of β-CTF and α-CTF by γ-secretase could have functional consequences is obvious. Indeed, AID-Stub1/CRL4CRBN complexes are released from membranes. This may, among other things, result into down-modulation of the APP-dependent ubiquitination of trans-membrane proteins. F, cleavage of APP-Stub1/CRL4CRBN, β-CTF-Stub1/CRL4CRBN, α-CTF-Stub1/CRL4CRBN, and AID-Stub1/CRL4CRBN by caspases could functionally separate the activities linked to the various ACR-Stub1 and ACR-CRL4CRBN complexes.

Article Snippet: In Vitro Ubiquitination Assay Pulldown samples were incubated in 50 m m Tris, pH 7.6, 5 m m MgCl 2 , 2 m m ATP, 0.6 m m DTT, with/without 40 ng of the E1 UBE1 (catalog no. E-305, lot 16114714, BostonBiochem, Cambridge, MA), 0.3 μg of the E2 UbcH5a/UBE2D1 (catalog no. E2-616, Lot 04201314C, BostonBiochem, Cambridge, MA), 1 μg of ubiquitin-FLAG (catalog no. U-211, Lot DBGI0215011, BostonBiochem), 1 μ m recombinant human HA ubiquitin aldehyde, C terminus (catalog no. U-556, lot 0AB03101C, BostonBiochem).

Techniques: Binding Assay, Functional Assay, Modification, Derivative Assay

Journal: Molecular cell

Article Title: ZNF598 and RACK1 Regulate Mammalian Ribosome-Associated Quality Control Function by Mediating Regulatory 40S Ribosomal Ubiquitylation

doi: 10.1016/j.molcel.2016.12.026

Figure Lengend Snippet: Key Resources Table

Article Snippet: Each reaction (20 µl) contained 1 µM Uba1, 1 µM UbcH5c, 20 mM Tris-Cl pH 7.6, 150 mM NaCl, 2 mM ATP, 5 mM MgCl 2 , 60 µM human recombinant ubiquitin (Boston Biochem), 5 ng FLAG-HA-ZNF598, and 5 µg of human ribosomal protein complexes isolated by 35% sucrose cushion ultracentrifugation.

Techniques: Western Blot, Protease Inhibitor, Recombinant, Bicinchoninic Acid Protein Assay, Mass Spectrometry, Sequencing, Software

Journal: iScience

Article Title: Evolutionary conserved regulation of TFEB stability by the E3 ubiquitin ligase WWP2 modulates response to stress in vivo

doi: 10.1016/j.isci.2025.111838

Figure Lengend Snippet:

Article Snippet: recombinant human 6His-Ube1 , RD System , Cat# E-305.

Techniques: Virus, Recombinant, Ubiquitin Proteomics, Luciferase, Mutagenesis, Reverse Transcription, SYBR Green Assay, Plasmid Preparation, Software, Lysis

Journal: Cell Death & Disease

Article Title: DNA damage and S phase-dependent E2F1 stabilization requires the cIAP1 E3-ubiquitin ligase and is associated with K63-poly-ubiquitination on lysine 161/164 residues

doi: 10.1038/cddis.2017.222

Figure Lengend Snippet: cIAP1 stabilizes and stimulates E2F1 in an E3-ubiquitin ligase activity-dependent manner. ( a, b and d ) E2F1 activity and expression in Hela cells ( a and b ) or in cIAP1 −/− /cIAP2 −/− MEFs ( d ) transfected with CCNE promoter-Firefly luciferase reporter plasmid, pCMV-3HA-E2F1, along with empty vector (Vector), cIAP1, cIAP1-H588A (devoid of E3-ubiquitin ligase activity) or cIAP1-F616A (lacking dimerization capacity) mutant encoding vector. HeLa cells were treated for 24 h with PYR-41 10–35 μ M before analysis ( a ). Upper panels: E2F1 transcriptional activity was assessed in gene luciferase experiments. Luciferase activity was normalized to β -galactosidase activity and expressed as fold induction of promoter stimulated by E2F1 alone. Mean±S.D. of at least three independent experiments. Statistical analysis performed using Student’s t -test. *** P <0.001, **0.001< P <0.01, *0.01< P <0.1, NS P >0.1. Lower panels: the expression of the constructs was analyzed by a western blot analysis. β -Actin or HSC70 are used as loading control. *Unspecific bands. ( c ) Ubiquitination assay performed in HeLa cells transfected with 3HA-E2F1, His-Ubiquitin encoding vectors, with an empty vector, cIAP1 or cIAP1 F616A (F/A) mutant constructs. Ubiquitinated proteins were pulled-down by using non-selective or K63-TUBEs and ubiquitinated E2F1 is revealed by using an anti-E2F1 antibody. ( e ) Western blot analysis of cIAP1 or 3HA-E2F1 expression in HeLa cells transfected with pCMV-3HA-E2F1 and with empty vector (Vector), cIAP1 or cIAP1-F616A mutant (dimerization defective mutant) encoding vector, treated or not with MG132 5 μ M overnight. ( f ) In vitro ubiquitination assay of GST-E2F1 fusion protein immobilized on gluthatione sepharose and incubated with ubiquitin, E1 and E2 recombinant proteins with or without recombinant cIAP1

Article Snippet: For the in vitro ubiquitination assay, GST-E2F1 fusion protein was produced in Escherichia coli by using the pGEX-E2F1 construct, immobilized on gluthatione sepharose beads (GE Healthcare) and incubated Sigma at 37 °C for 1 h with recombinant cIAP1 (R&D System), 100 nM of recombinant human E1 ubiquitin-activating enzyme UBE1 (Boston Biochem, Cambridge, MA, USA), 1.5 μ g of recombinant human E2 enzyme Ubc H5a/UBE2D1 (Boston Biochem), 0.2 mM of recombinant human ubiquitin (R&D Systems) in a buffer containing 75 mM Tris pH 8.2 mM DTT, 5 mM de MgCl 2 , 4 mM ATP.

Techniques: Ubiquitin Proteomics, Activity Assay, Expressing, Transfection, Luciferase, Plasmid Preparation, Mutagenesis, Construct, Western Blot, Control, In Vitro, Incubation, Recombinant

Journal: Cell Death & Disease

Article Title: DNA damage and S phase-dependent E2F1 stabilization requires the cIAP1 E3-ubiquitin ligase and is associated with K63-poly-ubiquitination on lysine 161/164 residues

doi: 10.1038/cddis.2017.222

Figure Lengend Snippet: cIAP1 overexpression revealed a K63 ubiquitination of E2F1 on lysine residue 161/164. ( a ) Schematic representation of E2F1 protein structure indicating the lysine residues and lysine clusters. ( a ) Cyclin A: CDK2-binding domain; DBD: DNA-binding domain, dimer: dimerization domain; MB: marked box: Rb: Rb-binding domain. ( b ) Gene luciferase experiments performed in HeLa cells transfected with CCNE promoter-Firefly luciferase reporter plasmid, pCMV-HA (vector), HA-tagged-E2F1 (one single or 3HA) or HA-E2F1 mutants in which indicated K have been mutated into R along with empty vector (vector) or cIAP1-encoding vector. Luciferase activity was normalized to β -galactosidase activity and expressed as fold induction of promoter stimulated by E2F1. Mean±S.D. of at least three independent experiments. Statistical analysis performed using Student’s t test. *** P <0.001, *0.01< P <0.1, NS P >0.1. The expression of the E2F1 constructs was checked by a western blot analysis (upper panel). β -Actin is used as loading control. ( c and d ) Ubiquitination assay performed in HeLa cells transfected with 3HA-E2F1 wt, K137R mutant or K161/164R mutant, His-Ubiquitin, with an empty vector or cIAP1 or cIAP1 F616A (F/A) mutant constructs. E2F1 is IP by using specific anti-E2F1 antibody and ubiquitination is revealed by using pan-ubiquitin antibody (FK2) ( c ), or ubiquitinated proteins were pulled-down by using K63-specific TUBEs and ubiquitinated E2F1 is revealed by using an anti-E2F1 antibody ( d ). ( e ) IP analysis of the interaction of cIAP1 with E2F1 wt or K161/164 R mutant. Hela cells were transfected with 3HA-E2F1 encoding constructs and cIAP1. cIAP1 or E2F1 were IP using anti-cIAP1 or anti-HA antibody and cIAP1-E2F1 interaction was revealed by a western blot analysis. KO: cell lysate from cIAP1 −/− KO MEFs was used to check nonspecific reactivity of the antibody

Article Snippet: For the in vitro ubiquitination assay, GST-E2F1 fusion protein was produced in Escherichia coli by using the pGEX-E2F1 construct, immobilized on gluthatione sepharose beads (GE Healthcare) and incubated Sigma at 37 °C for 1 h with recombinant cIAP1 (R&D System), 100 nM of recombinant human E1 ubiquitin-activating enzyme UBE1 (Boston Biochem, Cambridge, MA, USA), 1.5 μ g of recombinant human E2 enzyme Ubc H5a/UBE2D1 (Boston Biochem), 0.2 mM of recombinant human ubiquitin (R&D Systems) in a buffer containing 75 mM Tris pH 8.2 mM DTT, 5 mM de MgCl 2 , 4 mM ATP.

Techniques: Over Expression, Ubiquitin Proteomics, Residue, Binding Assay, Luciferase, Transfection, Plasmid Preparation, Activity Assay, Expressing, Construct, Western Blot, Control, Mutagenesis

Journal: Cell Death & Disease

Article Title: DNA damage and S phase-dependent E2F1 stabilization requires the cIAP1 E3-ubiquitin ligase and is associated with K63-poly-ubiquitination on lysine 161/164 residues

doi: 10.1038/cddis.2017.222

Figure Lengend Snippet: cIAP1 is required for DNA damage-induced stabilization of E2F1. ( a ) Western blot analysis of E2F1 and cIAP1 in U2OS cells transfected with control or cIAP1-siRNA and treated with indicated concentration of etoposide for 6 h. β -Actin was used as loading control. ( b ) Ubiquitination profile of E2F1 in HeLa cells transfected with cIAP1 siRNA or cIAP1-encoding construct and with 3HA-E2F1 and His-tagged ubiquitin wt. When indicated, cells were treated for 6 h with 10 μ M etoposide. E2F1 was IP using anti-HA antibody and ubiquitin revealed using K63-specific ubiquitin chain antibody (K63-Ub). The expression of the transgenes and the efficiency of siRNA were checked by a western blot analysis (input). β -Actin was used as loading control. ( c ) Ubiquitination assay performed in HeLa cells transfected with control or cIAP1 siRNA and with 3HA-E2F1 and His-tagged ubiquitin wt. When indicated, cells were treated for 6 h with 10 μ M etoposide. Ubiquitinated proteins were pulled-down by using K63 specific TUBEs and ubiquitinated E2F1 is revealed by using E2F1 antibody. ( d ) Western blot analysis of E2F1 in U2OS cells transfected with 3HA-E2F1 or 3HA-E2F1-K161/164R mutant and treated with indicated concentration of etoposide for 6 h. β -Actin was used as loading control. ( e ) Ubiquitination of E2F1 in HeLa cells transfected with pCMV-3HA-E2F1 or pCMV-3HA-E2F1 K161/164R, and with His-tagged ubiquitin wt, and then treated for 6 h with 10 μ M etoposide. E2F1 was IP using anti-HA antibody and ubiquitin revealed using K63-specific ubiquitin chain antibody (K63-Ub). The level of expression of E2F1 constructs has been adjusted in order to get equivalent ubiquitin level in both untreated samples. ( f ) U2OS cells were transfected with cIAP1 siRNA or treated with 17 nM GDC-0152 for 1 h, and then treated with 10 μ M etoposide for 48 h. tp73 mRNA expression was measured by RT-qPCR. UT: untreated cells. Results were normalized to hprt mRNA and expressed relatively to control untreated cells. Mean±S.D. of three independent experiments

Article Snippet: For the in vitro ubiquitination assay, GST-E2F1 fusion protein was produced in Escherichia coli by using the pGEX-E2F1 construct, immobilized on gluthatione sepharose beads (GE Healthcare) and incubated Sigma at 37 °C for 1 h with recombinant cIAP1 (R&D System), 100 nM of recombinant human E1 ubiquitin-activating enzyme UBE1 (Boston Biochem, Cambridge, MA, USA), 1.5 μ g of recombinant human E2 enzyme Ubc H5a/UBE2D1 (Boston Biochem), 0.2 mM of recombinant human ubiquitin (R&D Systems) in a buffer containing 75 mM Tris pH 8.2 mM DTT, 5 mM de MgCl 2 , 4 mM ATP.

Techniques: Western Blot, Transfection, Control, Concentration Assay, Ubiquitin Proteomics, Construct, Expressing, Mutagenesis, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: DNA damage and S phase-dependent E2F1 stabilization requires the cIAP1 E3-ubiquitin ligase and is associated with K63-poly-ubiquitination on lysine 161/164 residues

doi: 10.1038/cddis.2017.222

Figure Lengend Snippet: PRMT-mediated arginine methylation of E2F1 is required for regulation of E2F1 by cIAP1. ( a ) Western blot analysis of E2F1 and cIAP1 in HeLa cells transfected with 3HA-E2F1 and cIAP1-encoding constructs, and treated for 24 h with 100 μ M PRMT inhibitor AMI-1. HSC70 was used as loading control. ( b ) Ubiquitination of E2F1 in HeLa transfected with pCMV-3HA-E2F1, pCI-cIAP1, and with His-tagged ubiquitin wt, and then treated for 16 h with 50 μ M AMI-1±10 μ M etoposide for 6 h. E2F1 was IP using anti-E2F1 antibody and ubiquitin revealed using pan anti-ubiquitin antibody (FK2). The expression of the transgene is checked by western blot. β -Actin was used as loading control. ( c ) E2F1 transcriptional activity as measured by using a gene reporter luciferase assay performed in Hela cells transfected as in a in the presence of CCNE promoter-Firefly luciferase reporter plasmid and treated with increasing concentration of AMI-1 for 24 h. Results are expressed as average±S.D. from at least three experiments. Statistical analysis performed using Student’s t- test. *** P <0.001, **0.001< P <0.01, NS P >0.1. ( d ) Ubiquitination profile of E2F1 in HeLa transfected with Control (Co), PRMT-1 or PRMT-5-siRNA and pCMV-3HA-E2F1, pCI-cIAP1, and with His-tagged ubiquitin wt. E2F1 was IP using anti-HA antibody and ubiquitin revealed using pan anti-ubiquitin antibody (FK2). The expression of the transgene and the efficiency of siRNA were checked by western blot analysis. β -Actin was used as loading control

Article Snippet: For the in vitro ubiquitination assay, GST-E2F1 fusion protein was produced in Escherichia coli by using the pGEX-E2F1 construct, immobilized on gluthatione sepharose beads (GE Healthcare) and incubated Sigma at 37 °C for 1 h with recombinant cIAP1 (R&D System), 100 nM of recombinant human E1 ubiquitin-activating enzyme UBE1 (Boston Biochem, Cambridge, MA, USA), 1.5 μ g of recombinant human E2 enzyme Ubc H5a/UBE2D1 (Boston Biochem), 0.2 mM of recombinant human ubiquitin (R&D Systems) in a buffer containing 75 mM Tris pH 8.2 mM DTT, 5 mM de MgCl 2 , 4 mM ATP.

Techniques: Methylation, Western Blot, Transfection, Construct, Control, Ubiquitin Proteomics, Expressing, Activity Assay, Luciferase, Plasmid Preparation, Concentration Assay

Journal: Cell Death & Disease

Article Title: DNA damage and S phase-dependent E2F1 stabilization requires the cIAP1 E3-ubiquitin ligase and is associated with K63-poly-ubiquitination on lysine 161/164 residues

doi: 10.1038/cddis.2017.222

Figure Lengend Snippet: K63 ubiquitination of E2F1 is cell cycle regulated. ( a ) Western blot analysis of E2F1, cyclin A and cIAP1 in U2OS cells transfected with cyclin A siRNA±cIAP1 siRNA. HSC70 was used as loading control. ( b ) Ubiquitination profile of E2F1 in HeLa cells co-transfected with cyclin A siRNA±cIAP1 siRNA, then 24 h later with pCMV-3HA-E2F1 and His-tagged ubiquitin wt. E2F1 was IP using anti-E2F1 antibody and ubiquitin revealed using K63-specific ubiquitin chain antibody (K63-Ub). The expression of the transgenes was checked by a western blot analysis. β -Actin was used as loading control. ( c ) Western blot analysis of the expression of 3HA-E2F1 wt or K161/164 R mutant and cyclin A in HeLa cells transfected with cyclin A siRNA. β -Actin was used as loading control. ( d ) Ubiquitination profile of E2F1 in HeLa cells co-transfected with cyclin A siRNA, then 24 h later with pCMV-3HA-E2F1 or the K161/164 R mutant and His-tagged K63-only ubiquitin. E2F1 was IP using anti-E2F1 antibody and ubiquitin revealed using K63-specific ubiquitin chain antibody (K63-Ub). The expression of the transgenes was checked by a western blot analysis. β -Actin was used as loading control. The level of expression of E2F1 constructs has been adjusted in order to get equivalent ubiquitin level in both samples transfected with control siRNA. ( e ) Western blot analysis of cIAP1 E2F1, cyclin E, Rb, phospho (S780, S807/811) Rb in U2OS cells transfected with cIAP1-encoding construct. HSC70 was used as loading control. ( f ) Schematic representation of the regulation of E2F1. In the G1 phase of cell cycle, E2F1 is complexed to Rb. In the S phase, E2F1 is first methylated on arginine residue par PRMT, then K63-ubiquitinated on K161/164 in a cIAP1-dependent manner, leading to stabilization and activation of the protein. In the late S, cyclin A inhibits K63 ubiquitination of E2F1, binds to and phosphorylates E2F1 and promotes UPS-mediated degradation

Article Snippet: For the in vitro ubiquitination assay, GST-E2F1 fusion protein was produced in Escherichia coli by using the pGEX-E2F1 construct, immobilized on gluthatione sepharose beads (GE Healthcare) and incubated Sigma at 37 °C for 1 h with recombinant cIAP1 (R&D System), 100 nM of recombinant human E1 ubiquitin-activating enzyme UBE1 (Boston Biochem, Cambridge, MA, USA), 1.5 μ g of recombinant human E2 enzyme Ubc H5a/UBE2D1 (Boston Biochem), 0.2 mM of recombinant human ubiquitin (R&D Systems) in a buffer containing 75 mM Tris pH 8.2 mM DTT, 5 mM de MgCl 2 , 4 mM ATP.

Techniques: Ubiquitin Proteomics, Western Blot, Transfection, Control, Expressing, Mutagenesis, Construct, Methylation, Residue, Activation Assay

Journal: PLOS Pathogens

Article Title: Lumpy skin disease virus protein LSDV122 impairs IFN-I receptor complex formation to evade host innate immunity

doi: 10.1371/journal.ppat.1013871

Figure Lengend Snippet: A. The mRNA levels of ISG15 and ISG56 genes on lesion and adjacent non-lesion skin samples in vivo . Holstein cattle were intravenously injected with LSDV for 14 days, and then lesion and non-lesion skin samples from the same cattle were dissected for RT-qPCR analysis of mRNA levels of ISG15 and ISG56 genes. B. Effects of LSDV on IFN-β-induced transcription of ISGs. MDBK cells (5 × 10 5 ) were left un-infected or infected with wild-type LSDV (MOI = 1) for 12 hours and then treated with IFN-β (100 ng/ml, final concentration) for 6 hours followed by RT-qPCR analysis of mRNA levels of the indicated genes. C. Effects of LSDV on IFN-β-induced phosphorylation of STAT1 and STAT2. MDBK (5 × 10 5 ) cells were left uninfected or infected with wild-type LSDV (MOI = 1) for 12 hours and then treated with IFN-β (100 ng/ml) for the indicated times before immunoblotting analysis with the indicated antibodies. Data shown in A-B are mean ± SD (n = 11 in A and n = 3 in B) from one representative experiment. These experiments were repeated at least twice with similar results. ns nonsignificant, **P < 0.01 (unpaired t-test).

Article Snippet: At 5 and 10 hours post-infection, orbital blood samples were collected, and the levels of Isg15 and Cxcl10 in the serum were measured using ELISA kits (CSB-EL011843MO, CUSABIO; EK0736, BOSTER).

Techniques: In Vivo, Injection, Quantitative RT-PCR, Infection, Concentration Assay, Phospho-proteomics, Western Blot

Journal: PLOS Pathogens

Article Title: Lumpy skin disease virus protein LSDV122 impairs IFN-I receptor complex formation to evade host innate immunity

doi: 10.1371/journal.ppat.1013871

Figure Lengend Snippet: A. Effects of LSDV122-deficiency on LSDV-induced production of serum ISG15 and CXCL10. Seven-week-old C57BL/6 mice were intravenously injected with medium (mock, n = 4), LSDV (2.6 × 10 5 pfu each mouse, n = 6) or LSDVΔ122 (2.6 × 10 5 pfu each mouse, n = 5). The orbital blood of viral infected mice was collected at 5 and 10 hours post infection, and the orbital blood of mock injected mice was collected at 5 hours post injection for ELISA analysis of CXCL10 and ISG15 levels. B-D. Effects of LSDV122-deficiency on LSDV-induced transcription of antiviral genes in different tissues of mice. Seven-week-old C57BL/6 mice were intravenously injected with medium (mock, n = 4), LSDV (2.6 × 10 5 pfu each mouse, n = 6) or LSDVΔ122 (2.6 × 10 5 pfu each mouse, n = 5). Spleen (B), liver (C) and lung (D) of mice were collected at 5 or 10 hours post viral infection or 5 hours post mock injection for RT-qPCR analysis of mRNA levels of the indicated genes. Data shown in A-D are mean ± SD. These experiments were repeated at least twice with similar results. *P < 0.05; **P < 0.01 (unpaired t-test).

Article Snippet: At 5 and 10 hours post-infection, orbital blood samples were collected, and the levels of Isg15 and Cxcl10 in the serum were measured using ELISA kits (CSB-EL011843MO, CUSABIO; EK0736, BOSTER).

Techniques: Injection, Infection, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR