Journal: Glycobiology

Article Title: Inter domain linker region affects properties of CBM6 in GH5_34 arabinoxylanases and alters oligosaccharide product profile

doi: 10.1093/glycob/cwae048

Figure Lengend Snippet: A) SDS–PAGE of recombinant enzyme variants in lysate before purification. (M) – Precision plus protein unstained standards (bio-rad) molecular-mass marker, (1) Hh Xyn5A, (2) Hh-Hh-Ct , (3) Hh-Ct-Ct , (4) Ct-Ct-Hh , (5) Ct-Hh-Hh . B) SDS–PAGE of recombinant enzyme variants after purification and dialysis. (M) – Precision plus protein unstained standards (bio-rad) molecular-mass marker, (1) Hh Xyn5A, (2) Hh-Hh-Ct , (3) Hh-Ct-Ct , (4) Ct Xyn5A, (5) Ct-Ct-Hh , (6) Ct-Hh-Hh . C) Schematic representation of the domain organization of Hh Xyn5A and Ct Xyn5A enzyme variants in comparison with reference full-length multi-modular Hh Xyn5A-FULL ( Norlander et al. 2023 ) and Ct Xyn5A-FULL ( Montanier et al. 2011 ; Brás et al. 2011 ; Norlander et al. 2023 ) organisations. sp = signal peptide; GH = glycoside hydrolase; CBM = carbohydrate binding module; RIC = ricin-type beta-trefoil lectin domain; FN3 = fibronectin type 3 like domain; D = dockerin domain. Dotted lines surrounding domains that are included in the truncated two-domain variants used in this study. Numbering of the domains refers to the amino acid sequence interval. Indicated mutations are highlighted as bold dots.

Article Snippet: If domain splitting of recombinant proteins was observed, purified fragments were excised from stained SDS–PAGE gel and analysed applying MS (Bruker Daltonics).

Techniques: SDS Page, Recombinant, Purification, Marker, Comparison, Binding Assay, Sequencing

Journal: Journal of Interferon & Cytokine Research

Article Title: DHX15 Is a Coreceptor for RLR Signaling That Promotes Antiviral Defense Against RNA Virus Infection

doi: 10.1089/jir.2018.0163

Figure Lengend Snippet: DHX15 is an RIG-I-interacting protein that signals innate immune responses and required for antiviral immunity against RNA viruses. (A) HEK293 cells were cotransfected with FLAG-RIG-I and either a control Myc-vector or a plasmid expressing Myc-DHX15. Myc-DHX15 was immunoprecipitated and coprecipitating FLAG-RIG-I was analyzed by immunoblot analysis. Total cell lysate was analyzed directly by SDS-PAGE immunoblot as input. (B) The relative gene expression levels (%) of the genes, DHX15, RIG-I, and MAVS, in Huh 7 knockdown cells that stably express shRNA to DHX15, RIG-I, MAVS, or a control sequence not found in human or mouse cells (NTV) as measured by RT-PCR analysis. (C) Huh7 knockdown cells were cotransfected with ISG56/IFIT1 promoter-driven firefly luciferase reporter plasmid and a CMV promoter-driven Renilla luciferase reporter plasmid. Cells were mock-infected or infected with SeV for 24 h and then analyzed for dual luciferase activity. The results are expressed as average fold change in relative promoter activity of SeV-infected cells over mock-infected cells. (D) Huh7 knockdown cells were cotransfected with an IFN-β promoter-driven firefly luciferase reporter plasmid, a CMV promoter-driven Renilla luciferase reporter plasmid, and either a vector control plasmid or a plasmid that ectopically expressed either Flag-DHX15 or the Flag-DHX15 AAAA mutant. Cells were mock-infected or infected with SeV for 24 h and then analyzed for dual luciferase activity. The results are expressed as average fold change in relative promoter activity of SeV-infected cells over mock-infected cells. (E) Representative immunoblot showing expression of Flag-DHX15 and Flag-DHX15 AAAA relative to a tubulin loading control using cell lysates generated in parallel with (D). (F) Huh7 or (G) PMA-differentiated THP-1 knockdown cells were mock-infected or infected with SeV. Total cellular RNA was collected at 0, 6, 12, 18, and 24 h postinfection and analyzed by RT-PCR for various innate immune, cytokine, and IFN genes, normalized to GAPDH. Graphs show average fold induction over mock-infected controls with error bars showing standard deviation. (H) Huh7 knockdown cells were mock-infected or infected with SeV and total cellular RNA collected at 0, 6, 12, 18, and 24 h postinfection and analyzed by RT-PCR for SeV genomic RNA against a standard. Results show average SeV RNA copy number from 3 independent experiments with error bars showing standard deviation. (I) Huh7 knockdown cells were mock-infected or infected with VSV at an MOI of 0.01 for 12 h. Infectious virus particle numbers in the cell culture supernatant were measured by plaque assay on Vero cells at 24 h postinfection. Results show average infectious virus particle numbers calculated per milliliter supernatant from 3 independent experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P ≤ 0.0001. IFN, interferon; ISG, interferon-stimulated genes; MAVS, mitochondrial antiviral signaling; MOI, multiplicity of infection; NTV, nontargeting vector; PMA, phorbol myristate acetate; RIG-1, retinoic acid inducible gene-I; RT-PCR, real-time polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate/polyacrylamide gel electrophoresis; SeV, Sendai virus; shRNA, short hairpin RNA; VSV, vesicular stomatitis virus.

Article Snippet: The DHX15 recombinant protein was expressed in Escherichia coli using expression vector E3 and purified from inclusion bodies to ∼95% purity as estimated by densitometric analysis of the Coomassie blue-stained SDS-PAGE gel (GenScript).

Techniques: Plasmid Preparation, Expressing, Immunoprecipitation, Western Blot, SDS Page, Stable Transfection, shRNA, Sequencing, Reverse Transcription Polymerase Chain Reaction, Luciferase, Infection, Activity Assay, Mutagenesis, Generated, Standard Deviation, Cell Culture, Plaque Assay, Real-time Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis

Journal: Journal of Interferon & Cytokine Research

Article Title: DHX15 Is a Coreceptor for RLR Signaling That Promotes Antiviral Defense Against RNA Virus Infection

doi: 10.1089/jir.2018.0163

Figure Lengend Snippet: DHX15 interacts with RIG-I, MDA5, and MAVS to initiate innate immune responses upstream of IFNAR signaling. (A) Huh7 knockdown cells were cotransfected with an ISG56/IFIT1 promoter-driven firefly and CMV promoter-driven Renilla luciferase reporter plasmids. Cells were mock-treated or treated with exogenous IFN-β in the media for 24 h and analyzed for dual luciferase activity. Results are expressed as relative luciferase activity. (B) Huh7 cells were treated with IFN-β (100 IU/mL). Cells were collected at 2, 12, or 24 h and the relative expression of the ISG56/IFIT1 and DHX15 genes was measured by RT-PCR, normalized to GAPDH and expressed as fold-induction over the mock-treated control. (C) HEK293 cells were mock-infected or infected with SeV for 7 h. Endogenous DHX15 was immunoprecipitated from cell lysates. DHX15 and coprecipitating proteins were eluted using SDS-PAGE sample buffer and analyzed by Western blot analysis. Total cell lysate was analyzed directly as input. (D) Huh7 knockdown cells (NTV control or DHX15 knockdown cells) were cotransfected with the IFN-β promoter-driven firefly luciferase reporter plasmid, a CMV promoter-driven Renilla luciferase reporter plasmid, and either a vector control plasmid or a plasmid that ectopically expressed constitutively active signaling factors of the RLR pathway: N-RIG, N-MDA5, MAVS, TBK-1, IKKɛ, and IRF3-5D. Lysates were collected 24 h after transfection and analyzed for dual luciferase reporter activity. The results are expressed as relative luciferase activity. Error bars show standard deviations. (E) HEK293 cells were cotransfected with FLAG-DHX15 along with an Myc-vector control or plasmids expressing Myc-RIG-I, Myc-MDA5, or Myc-MAVS. Cells were either mock-infected or infected with SeV for 24 h. Myc-tagged proteins were immunoprecipitated from cell lysates and analyzed by immunoblot for Myc-tag proteins and coprecipitating FLAG-DHX15 protein. Total cell lysate was analyzed directly by Western blot as input. (F) Huh7 knockdown cells were transfected with a vector control or a plasmid expressing NMDA5 (MDA5 CARDs alone) to turn on MDA5-specific innate immune signaling. Total RNA was collected 18 h post-transfection and analyzed by RT-PCR for expression of various IFN, cytokine, and innate immune genes. The graphs show average fold gene induction with NMDA5 overexpression relative to vector transfection controls. (G) Huh7 knockdown cells were mock-infected or infected with EMCV at an MOI of 0.01 for 18 h. Infectious virus particles in the cell culture supernatant were measured by plaque assay on Vero cells at 24 h postinfection. Results show average infectious virus particle numbers calculated per milliliter cell culture supernatant from 3 independent experiments. ns, P > 0.05; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****p ≤ 0.0001. CARDs, caspase activation and recruitment domains; EMCV, encephalomyocarditis virus; IRF3, interferon regulatory factor 3; MDA5, melanoma differentiation-associated protein 5; RLR, RIG-I-like receptor.

Article Snippet: The DHX15 recombinant protein was expressed in Escherichia coli using expression vector E3 and purified from inclusion bodies to ∼95% purity as estimated by densitometric analysis of the Coomassie blue-stained SDS-PAGE gel (GenScript).

Techniques: Luciferase, Activity Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Infection, Immunoprecipitation, SDS Page, Western Blot, Plasmid Preparation, Transfection, Over Expression, Cell Culture, Plaque Assay, Activation Assay

Journal: Journal of Interferon & Cytokine Research

Article Title: DHX15 Is a Coreceptor for RLR Signaling That Promotes Antiviral Defense Against RNA Virus Infection

doi: 10.1089/jir.2018.0163

Figure Lengend Snippet: N-terminus of DHX15 binds RIG-I CARDs. (A) Schematic of RIG-I protein and conserved motifs. Also represented are the various RIG-I truncation mutants we have created to define interactions with DHX15 by immunoprecipitation, the results of which are summarized on the right. (B) HEK293 cells were cotransfected with either a control Myc vector or a plasmid expressing Myc-DHX15 along with Flag-RIG-I constructs that express full-length RIG-I (aa 1–925), the constitutively active N-RIG (aa 1–228), or the dominant negative C-RIG (aa 219–925). Cell lysates were prepared from which Myc-DHX15 was immunoprecipitated using an anti-Myc mouse monoclonal antibody. Coprecipitating proteins were eluted from the beads in SDS-PAGE sample buffer and analyzed by Western blot analysis for various Flag-tagged RIG-I proteins. Twenty micrograms of total cell lysate was analyzed on SDS-PAGE Western blot to confirm protein expression and labeled as input. Asterisk denotes a nonspecific band detected by immunoblot analysis. (C) Schematic of DHX15 protein and conserved motifs. Also represented are the various DHX15 truncation mutants we have created to define interactions with RIG-I by immunoprecipitation, the results of which are summarized on the right. (D) HEK293 cells were cotransfected with FLAG-RIG-I along with either a control Myc vector or a plasmid expressing Myc-tagged full-length DHX15 (aa 1–795), Myc-tagged DHX15 mutants with N-terminal truncations aa 147–795 and 519–795, Myc-tagged DHX15 mutants with C-terminal truncations aa 1–661, 1–340, and 1–293, or Myc-tagged DHX15 with alanine mutations in its DEAH box (AAAA). Cell lysates were prepared from which Myc-tagged proteins were immunoprecipitated using an anti-Myc mouse monoclonal antibody. Coprecipitating proteins were eluted from the beads in SDS-PAGE sample buffer and analyzed by immunoblot analysis for Myc-DHX15 and FLAG-RIG-I. Twenty micrograms of total cell lysate was analyzed on SDS-PAGE immunoblot to confirm expression of the various constructs as input. Asterisks denote nonspecific bands as detected by immunoblot analysis.

Article Snippet: The DHX15 recombinant protein was expressed in Escherichia coli using expression vector E3 and purified from inclusion bodies to ∼95% purity as estimated by densitometric analysis of the Coomassie blue-stained SDS-PAGE gel (GenScript).

Techniques: Immunoprecipitation, Plasmid Preparation, Expressing, Construct, Dominant Negative Mutation, SDS Page, Western Blot, Labeling