rig i ddx58 rabbit polyclonal 25068 1 ap  (Proteintech)

Journal: Cell Reports

Article Title: CK2 phosphorylation of CMTR1 promotes RNA cap formation and influenza virus infection

doi: 10.1016/j.celrep.2024.114405

Figure Lengend Snippet:

Article Snippet: Anti-IFIT3 rabbit polyclonal , Proteintech , 15201-1-AP; RRID:AB_2248738.

Techniques: Recombinant, Transduction, RNA Sequencing, Software

Journal: Journal of Virology

Article Title: Porcine deltacoronavirus nsp5 antagonizes type I interferon signaling by cleaving IFIT3

doi: 10.1128/jvi.01682-23

Figure Lengend Snippet: PDCoV and poly(I:C) induced IFIT3 expression. ( A ) Heatmap showing the fold changes in the expression of genes in PDCoV-infected ST cells compared with mock-infected ST cells at 24 h. ( B ) Volcano plot showing the global differentially expressed genes in PDCoV-infected ST cells compared with mock-infected ST cells at 24 h. ( C ) pXJ40-HA-vector, pXJ40-HA-pIFIT3, IFIT3 siRNA, and negative control siRNA were transfected into ST cells. After 24 h of transfection, the cells were infected with PDCoV (MOI = 1) for 6, 12, and 24 h and were then harvested for Western blot analysis. Relative expression levels in the pXJ40-HA-vector, pXJ40-HA-pIFIT3, IFIT3 siRNA, and negative control siRNA groups were determined and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression using ImageJ. ( D and E ) HEK-293T and ST cells were treated with poly(I:C) (2 µg/mL) for 0, 4, 8, 12, or 24 h. And HEK-293T and ST cells were infected with SeV for 0, 4, 8, 12, or 24 h. The cells were lysed and collected, and the IFIT3 mRNA levels were then measured by RT-qPCR. The protein expression levels of IFIT3 and GAPDH were measured by Western blotting. All data are reported as means ± SDs. For all experiments, ** P < 0.01, and *** P < 0.001 were considered to indicate statistical significance.

Article Snippet: After three washes with PBS, the cells were blocked with PBS containing 3% bovine serum albumin (Solarbio, China) for 1 h and were then incubated separately with a rabbit anti-IFIT3 polyclonal antibody (Proteintech, China; 1:200) and a mouse anti-Flag tag antibody (Bioss, China; 1:200) for 2 h at room temperature or overnight at 4°C.

Techniques: Expressing, Infection, Plasmid Preparation, Negative Control, Transfection, Western Blot, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Porcine deltacoronavirus nsp5 antagonizes type I interferon signaling by cleaving IFIT3

doi: 10.1128/jvi.01682-23

Figure Lengend Snippet: IFIT3 activates the type I IFN signaling pathway. ( A ) HEK-293T cells were cotransfected with the IFN-β Luc and pRL-TK plasmids, along with 0.5, 1, or 1.5 µg of the pXJ40-HA-pIFIT3 expression plasmid for 30 h. The cells were lysed and subjected to a dual-luciferase assay. ( B ) HEK-293T cells were transfected with 0.5, 1, or 1.5 µg of the pXJ40-HA-pIFIT3 for 30 h. The cells were collected, and the IFN-β mRNA level was measured by RT-qPCR. ( C ) HEK-293T cells were transfected with 0.5, 1, or 1.5 µg of the pXJ40-HA-pIFIT3. After 30 h, the cells were collected, and the ISG56, ISG54, IFIT5, RIG-I, MDA-5, MAVs, IκBα , IRF3, and NF-κB mRNA levels were measured by RT-qPCR. ( D ) pXJ40-HA-vector, pXJ40-HA-pIFIT3, IFIT3 siRNA, and negative control siRNA were transfected into HEK-293T cells. After 30 h of transfection, the cells were collected and analyzed by Western blotting with anti-P65, anti-phospho-P65, anti-IRF3, anti-phospho-IRF3, anti-STAT1, anti-phospho-STAT1, anti-TBK1, anti-phospho-TBK1, anti-IFIT3, and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies. Relative levels of P65, phospho-P65, IRF3, phospho-IRF3, STAT1, phospho-STAT1, TBK1, and phospho-TBK1 were quantified and normalized to the GAPDH expression level using ImageJ. All data are reported as the means ± SDs. For all experiments, * P < 0.05, ** P < 0.01, and *** P < 0.001 were considered to indicate statistical significance. ns, nonsignificant difference.

Article Snippet: After three washes with PBS, the cells were blocked with PBS containing 3% bovine serum albumin (Solarbio, China) for 1 h and were then incubated separately with a rabbit anti-IFIT3 polyclonal antibody (Proteintech, China; 1:200) and a mouse anti-Flag tag antibody (Bioss, China; 1:200) for 2 h at room temperature or overnight at 4°C.

Techniques: Expressing, Plasmid Preparation, Luciferase, Transfection, Quantitative RT-PCR, Negative Control, Western Blot

Journal: Journal of Virology

Article Title: Porcine deltacoronavirus nsp5 antagonizes type I interferon signaling by cleaving IFIT3

doi: 10.1128/jvi.01682-23

Figure Lengend Snippet: PDCoV nsp5 inhibits the SeV-induced type I IFN signaling pathway. ( A ) ST cells were infected with the PDCoV (MOI = 1). After 12 h, the cells were further infected or mock infected with SeV or mock infected for 12 h. Cells were collected at 12 h post infection, and the IFN-β, RIG-I, MDA-5, ISG56, ISG54, IFIT3, ISG15, IRF9, RSAD2, and CXCL10 mRNA levels were measured by RT-qPCR. ( B ) HEK-293T cells were cotransfected with the IFN-β Luc and pRL-TK plasmids, along with 1.2 µg of pCAGGS-flag-nsp5 or an empty vector. After 24 h, the cells were further infected or mock infected with SeV or mock infected for 12 h. The cells were lysed and subjected to a dual-luciferase assay. ( C ) ST cells were transfected with 1.2 µg of pCAGGS-flag-nsp5 or an empty vector. After 24 h of initial transfection, the cells were further infected or mock infected with SeV or mock infected. The cells and supernatants were collected at 12 h post infection, and the IFN-β, ISG56, ISG54, IFIT3, and IFIT5 mRNA levels were measured by RT-qPCR. ( D ) HEK-293T cells were transfected with the pCAGGS-flag-nsp5 for 24 h and stimulated with SeV for 12 h. Cell lysates were analyzed by Western blotting with anti-P65, anti-phospho-P65, anti-IRF3, anti-phospho-IRF3, anti-STAT1, anti-phospho-STAT1, anti-TBK1, anti-phospho-TBK1, anti-IFIT3, and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies. Relative levels of P65, phospho-P65, IRF3, phospho-IRF3, STAT1, phospho-STAT1, TBK1, and phospho-TBK1 were quantified and normalized to the GAPDH expression level using ImageJ. All data are reported as the means ± SDs. For all experiments, * P < 0.05, ** P < 0.01, and *** P < 0.001 were considered to indicate statistical significance.

Article Snippet: After three washes with PBS, the cells were blocked with PBS containing 3% bovine serum albumin (Solarbio, China) for 1 h and were then incubated separately with a rabbit anti-IFIT3 polyclonal antibody (Proteintech, China; 1:200) and a mouse anti-Flag tag antibody (Bioss, China; 1:200) for 2 h at room temperature or overnight at 4°C.

Techniques: Infection, Quantitative RT-PCR, Plasmid Preparation, Luciferase, Transfection, Western Blot, Expressing

Journal: Journal of Virology

Article Title: Porcine deltacoronavirus nsp5 antagonizes type I interferon signaling by cleaving IFIT3

doi: 10.1128/jvi.01682-23

Figure Lengend Snippet: PDCoV nsp5 targets IFIT3 for cleavage. ( A ) HEK-293T cells were cultured in 6-well plates and cotransfected with the PDCoV nsp5 expression plasmid or empty vector along with 1.2µg of the HA-tagged IFIT1, IFIT2, IFIT3, IFIT5, RSAD2, USP18, OAS1, or TRAF2 expression plasmid. After 30h, the cells were lysed and analyzed by Western blotting with an anti-HA antibody. ( B–D ) HEK-293T, LLC-PK1, and IPEC-J2 cells were cotransfected with pXJ40-HA-pIFIT3 and various amounts of pCAGGS-flag-nsp5. After 30 h, the cells were lysed for Western blotting. The cleavage products are labeled “Cp.” ( E ) ST cells were transfected with 1.2 µg/well pXJ40-HA-pIFIT3. After 24 h of transfection, the ST cells were infected with PDCoV at MOIs of 1, 2, and 3. After 24 h, the cells were lysed for Western blotting. ( F ) ST cells were infected with PDCoV (MOI = 1), and the cells and supernatants were collected at 0, 6, 12, and 24 h for analysis by Western blot analysis. ( G ) IPEC-J2 cells were infected with PDCoV (MOI = 1), and the cells and supernatants were collected at 0, 12, 24, and 36 h for analysis by Western blot analysis. ( H ) HEK-293T cells were transfected with expression constructs encoding pCAGGS-flag-nsp5 and pXJ40-HA-pIFIT3. The cells were lysed 30 h after transfection and subjected to immunoprecipitation with an anti-FLAG antibody or anti-HA antibody. The whole-cell lysates (WCLs) and immunoprecipitation (IP) complexes were analyzed by immunoblotting using anti-FLAG, anti-HA, or anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies. ( I ) ST cells were transfected with PDCoV nsp5 and nsp13. After 24 h, the cells were fixed and then stained with a rabbit monoclonal antibody against IFIT3 and a mouse anti-flag tag antibody prior to incubation with an Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (green) or Alexa Fluor 594-conjugated goat anti-rabbit IgG antibody (red). Nuclei were stained with DAPI (blue).

Article Snippet: After three washes with PBS, the cells were blocked with PBS containing 3% bovine serum albumin (Solarbio, China) for 1 h and were then incubated separately with a rabbit anti-IFIT3 polyclonal antibody (Proteintech, China; 1:200) and a mouse anti-Flag tag antibody (Bioss, China; 1:200) for 2 h at room temperature or overnight at 4°C.

Techniques: Cell Culture, Expressing, Plasmid Preparation, Western Blot, Labeling, Transfection, Infection, Construct, Immunoprecipitation, Staining, FLAG-tag, Incubation

Journal: Journal of Virology

Article Title: Porcine deltacoronavirus nsp5 antagonizes type I interferon signaling by cleaving IFIT3

doi: 10.1128/jvi.01682-23

Figure Lengend Snippet: PDCoV nsp5 cleaves IFIT3 via its protease activity. ( A ) HEK-293T cells were cotransfected with plasmids expressing the wild-type PDCoV nsp5 or one of protease-defective mutants (C144A and H41A) and the pXJ40-HA-pIFIT3. After 30 h, the cells were lysed, and supernatants were used for Western blotting. ( B ) HEK-293T cells were cotransfected with pXJ40-HA-pIFIT3 and pCAGGS-flag-nsp5. After 24 h, the cells were mock treated or treated with PF-00835231 (10 µM). After 12 h, the cells were lysed for Western blotting. ( C ) HEK-293T cells were cotransfected with plasmids expressing the wild-type PDCoV nsp5 and pXJ40-HA-pIFIT3. After 24 h, the cells were treated with MG132 (10 µM), Z-VAD-FMK (25 µM), or 3-MA (10 µM) for 12 h. Cell lysates were prepared and analyzed by Western blotting. ( D ) HEK-293T and LLC-PK1 cells were cotransfected with the IFN-β-Luc and pRL-TK plasmids, along with 1.2 µg of pCAGGS-flag-nsp5, one of its protease-defective mutants (C144A, H41A, or DM-41-144) , or empty vector. After 30 h, the cells were lysed and subjected to a dual-luciferase assay. ( E–G ) HEK-293T cells seeded in a 12-well plate were transfected with equal amounts of plasmids encoding PDCoV nsp5 or one of its protease-defective mutants (C144A, H41A, and DM-41-144) for 24 h and were then infected with SeV for 12 h. The cell supernatants were then collected, treated with UV irradiation for 30 min, and added to a new 12-well plate of HEK-293T cells for 24 h. The IFN-treated cells were then inoculated with VSV-GFP for 12 h, and GFP expression was detected by flow cytometry, immunofluorescence staining, and Western blotting. Relative expression levels of GFP were quantified and normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression level using ImageJ. All data are reported as the means ± SDs. For all experiments, ** P < 0.01, and *** P < 0.001 were considered to indicate statistical significance. ns, nonsignificant difference.

Article Snippet: After three washes with PBS, the cells were blocked with PBS containing 3% bovine serum albumin (Solarbio, China) for 1 h and were then incubated separately with a rabbit anti-IFIT3 polyclonal antibody (Proteintech, China; 1:200) and a mouse anti-Flag tag antibody (Bioss, China; 1:200) for 2 h at room temperature or overnight at 4°C.

Techniques: Activity Assay, Expressing, Western Blot, Plasmid Preparation, Luciferase, Transfection, Infection, Irradiation, Flow Cytometry, Immunofluorescence, Staining

Journal: Journal of Virology

Article Title: Porcine deltacoronavirus nsp5 antagonizes type I interferon signaling by cleaving IFIT3

doi: 10.1128/jvi.01682-23

Figure Lengend Snippet: PDCoV nsp5 recognizes and cleaves IFIT3 at residue Q406. ( A ) HEK-293T cells were transfected with expression constructs encoding pXJ40-HA-pIFIT3, the pIFIT3 1-293 truncated mutant, and pIFIT3 1-371 truncated mutant and were collected after 30 h for Western blotting. ( B ) Sequence logo of the polyprotein junctions that are cleaved by PDCoV nsp5. An amino acid sequence logo of the substrate was generated by WebLogo version 3. ( C ) Schematic representation of pIFIT3 and its mutation sites. ( D ) HEK-293T cells were cotransfected with the pCAGGS-flag-nsp5 along with expression constructs encoding wild-type pIFIT3 or pIFIT3 mutants (pIFIT3-Q384A, pIFIT3-Q406A, and pIFIT3-Q436A). The cells were then lysed after 30 h and analyzed by Western blotting. ( E ) HEK-293T cells were co-transfected with the PDCoV nsp5 expression plasmid along with wild-type IFIT3 or pIFIT3-Q406A and collected after 30 h for Western blotting. ( F and G ) ST cells were transfected with empty vector, pIFIT3, pIFIT3-Q406A, pIFIT3 1-406 , or pIFIT3 406-510 . After 24 h, the cells were infected with PDCoV (MOI = 1) for 24 h and were then lysed and collected for analysis by Western blotting and RT-qPCR. All data are reported as the means ± SDs. For all experiments, * P < 0.05 and *** P < 0.001 were considered to indicate statistical significance. ns, nonsignificant difference.

Article Snippet: After three washes with PBS, the cells were blocked with PBS containing 3% bovine serum albumin (Solarbio, China) for 1 h and were then incubated separately with a rabbit anti-IFIT3 polyclonal antibody (Proteintech, China; 1:200) and a mouse anti-Flag tag antibody (Bioss, China; 1:200) for 2 h at room temperature or overnight at 4°C.

Techniques: Residue, Transfection, Expressing, Construct, Mutagenesis, Western Blot, Sequencing, Generated, Plasmid Preparation, Infection, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Porcine deltacoronavirus nsp5 antagonizes type I interferon signaling by cleaving IFIT3

doi: 10.1128/jvi.01682-23

Figure Lengend Snippet: The efficiency of Nsp5-mediated cleavage of IFIT3 differs across coronaviruses. ( A ) Porcine, human, monkey, canine. and bovine sequences of IFIT3 were aligned with DNAMAN8. ( B ) HEK-293T cells were cultured in 12-well plates and cotransfected with pXJ40-HA-pIFIT3 and a plasmid encoding the nsp5 of SADS-CoV, SARS-CoV-2, SARS-CoV, MERS-CoV, or PDCoV. After 30 h, the cells were lysed and analyzed by Western blotting. ( C ) HEK-293T cells were cotransfected with PDCoV nsp5 and pXJ40-HA-B-IFIT3 (bovine), pXJ40-HA-M-IFIT3 (monkey), pXJ40-HA-D-IFIT3 (canine), pXJ40-HA-P-IFIT3 (porcine), and pXJ40-HA-H-IFIT3 (human). After 30 h, the cells were lysed and analyzed by Western blotting.

Article Snippet: After three washes with PBS, the cells were blocked with PBS containing 3% bovine serum albumin (Solarbio, China) for 1 h and were then incubated separately with a rabbit anti-IFIT3 polyclonal antibody (Proteintech, China; 1:200) and a mouse anti-Flag tag antibody (Bioss, China; 1:200) for 2 h at room temperature or overnight at 4°C.

Techniques: Cell Culture, Plasmid Preparation, Western Blot

Journal: Journal of Virology

Article Title: Porcine deltacoronavirus nsp5 antagonizes type I interferon signaling by cleaving IFIT3

doi: 10.1128/jvi.01682-23

Figure Lengend Snippet: PDCoV nsp5 inhibits host innate immunity by directly cleaving IFIT3. Mechanistic diagram showing the antagonism of the IFIT3-mediated type I IFN signaling pathway by Nsp5 of PDCoV. The schematic was drawn using the Biorender website.

Article Snippet: After three washes with PBS, the cells were blocked with PBS containing 3% bovine serum albumin (Solarbio, China) for 1 h and were then incubated separately with a rabbit anti-IFIT3 polyclonal antibody (Proteintech, China; 1:200) and a mouse anti-Flag tag antibody (Bioss, China; 1:200) for 2 h at room temperature or overnight at 4°C.

Techniques:

Journal: Journal of Virology

Article Title: Porcine deltacoronavirus nsp5 antagonizes type I interferon signaling by cleaving IFIT3

doi: 10.1128/jvi.01682-23

Figure Lengend Snippet: Sequences of the primers and siRNAs

Article Snippet: After three washes with PBS, the cells were blocked with PBS containing 3% bovine serum albumin (Solarbio, China) for 1 h and were then incubated separately with a rabbit anti-IFIT3 polyclonal antibody (Proteintech, China; 1:200) and a mouse anti-Flag tag antibody (Bioss, China; 1:200) for 2 h at room temperature or overnight at 4°C.

Techniques: