p irf3  (Bioss)

Journal: Communications Biology

Article Title: Bmp8a is an essential positive regulator of antiviral immunity in zebrafish

doi: 10.1038/s42003-021-01811-0

Figure Lengend Snippet: a , b , e , f Expression of irf3 , irf7 , and tbk1 mRNA after transfected with 2 μg bmp8a or empty vector in ZFL ( a , b ) or EPC ( e , f ) cells. The cells were collected at 36 h ( a , e ) or 48 h ( b , f ) post-transfection. c , d , g , h Expression of irf3 , irf7 , and tbk1 mRNA after transfected with 2 μg bmp8a or empty vector in ZFL ( c , d ) or EPC ( g , h ) cells for 24 h, followed by infection with GCRV for another 24 h ( c , g ) or 36 h ( d , h ). i – l Expression of irf3 , irf7 , and tbk1 mRNA after bmp8a knockdown in ZFL cells. The cells were collected at 36 h ( i ) and 48 h ( j ) post-knockdown or at 24 h ( k ) and 36 h ( l ) post-infected with GCRV. m , o Immunoblot analysis of phosphorylated (p-) Tbk1 and Irf3 after transfected with 2 μg bmp8a or empty vector in ZFL ( m ) or EPC ( o ) cells. The cells were collected at 36 or 48 h post-transfection for Immunoblot analysis. n , p Immunoblot analysis of phosphorylated (p-) Tbk1 and Irf3 after transfected with 2 μg bmp8a or empty vector in ZFL ( n ) or EPC ( p ) cells for 24 h, followed by infection with GCRV for another 24 or 36 h. q , r Immunoblot analysis of phosphorylated (p-) TBK1 and IRF3 after bmp8a knockdown in ZFL cells. The cells were collected at 36 and 48 h post-knockdown or at 24 and 36 h post-infected with GCRV. s – u EPC cells were cotransfected with IFN-φ1pro-luc (200 ng, s ), IFN-φ3pro-luc (200 ng, t ) or EPC IFNpro-luc (200 ng, u ), and bmp8a (100 ng) together with each of the dominant negative plasmids including tbk1–K38M (100 ng), irf3DN (100 ng) and irf7DN (100 ng). At 48 h post-transfection, the cells were collected for luciferase assays. Renilla luciferase was used as the internal control. v – y Expression of irf3 , irf7 , and tbk1 mRNA in the liver, kidney, intestine, and spleen from WT or bmp8a −/− zebrafish injected i.p. with 50 µl of GCRV (10 8 TCID 50 per ml). The expression of zebrafish actb1 or EPC actin was used as an internal control for the qRT-PCR. Data were from three independent experiments and were analyzed by Student’s t -test (two-tailed) for comparison of two groups or one-way ANOVA followed by Games–Howell post hoc tests for comparison of multiple groups. All data were presented as mean ± SD (** p < 0.01, *** p < 0.001).

Article Snippet: Antibodies from Bioss: IRF3 (1:1000, #bs-2993R), p-IRF3 (Ser386) (1:1000, #bsm-52170R), p38MAPK (1:1000, #bs-0637R), p-p38MAPK (Thr180 + Tyr182) (1:1000, #bs-2210R), Actin (1:2000, #bs-0061R).

Techniques: Expressing, Transfection, Plasmid Preparation, Infection, Western Blot, Dominant Negative Mutation, Luciferase, Injection, Quantitative RT-PCR, Two Tailed Test

Journal: Communications Biology

Article Title: Bmp8a is an essential positive regulator of antiviral immunity in zebrafish

doi: 10.1038/s42003-021-01811-0

Figure Lengend Snippet: a , b Expression of alk2 , alk3 , alk6a , bmpr2a , bmpr2b , actr2a , and actr2b mRNA in ZFL cells stimulated with poly(I:C) (2 μg/ml, a ) or GCRV (5 × 10 4 TCID 50 per ml, b ) for 48 h. c – e Expression of ifnφ1 ( c ) and ifnφ3 ( d ) mRNA in ZFL cells or EPC ifn ( e ) in EPC cells which were transfected with 2 μg of alk2, alk3, alk6a, bmpr2a, bmpr2b, actr2a, actr2b or empty vector for 48 h. f , h Expression of irf3 , irf7 , tbk1 , ifn (or ifnφ1 and ifnφ3 ), and mx mRNA after transfected with 2 μg of pcDNA3.1-alk6a or empty vector in ZFL ( f ) or EPC ( h ) cells for 48 h. g , i Expression of irf3 , irf7 , tbk1 , ifn (or ifnφ1 and ifnφ3 ), and mx mRNA after transfected with 2 μg of of pcDNA3.1-alk6a or empty vector in ZFL ( g ) or EPC ( i ) cells for 24 h, followed by infection with GCRV for another 36 h. j Schematic drawing of the alk6a-ΔGS mutation that the GS domain of Alk6a was deleted. k , m Expression of irf3 , irf7 , tbk1 , ifn (or ifnφ1 and ifnφ3 ), and mx mRNA after transfected with 2 μg pcDNA3.1-alk6a-ΔGS or empty vector in ZFL ( k ) or EPC ( m ) cells for 48 h. l , n Expression of irf3 , irf7 , tbk1 , ifn (or ifnφ1 and ifnφ3 ), and mx mRNA after transfected with 2 μg pcDNA3.1-alk6a-ΔGS or empty vector in ZFL ( l ) or EPC ( n ) cells for 24 h, followed by infection with GCRV for another 36 h. The expression of zebrafish actb1 or EPC actin was used as an internal control for the qRT-PCR. Data were from three independent experiments and were analyzed by Student’s t -test (two-tailed) for comparison of two groups or one-way ANOVA followed by Games–Howell posthoc tests for comparison of multiple groups. All data were presented as mean ± SD (* p < 0.05, ** p < 0.01, and *** p < 0.001, ns means no significant difference).

Article Snippet: Antibodies from Bioss: IRF3 (1:1000, #bs-2993R), p-IRF3 (Ser386) (1:1000, #bsm-52170R), p38MAPK (1:1000, #bs-0637R), p-p38MAPK (Thr180 + Tyr182) (1:1000, #bs-2210R), Actin (1:2000, #bs-0061R).

Techniques: Expressing, Transfection, Plasmid Preparation, Infection, Mutagenesis, Quantitative RT-PCR, Two Tailed Test

Journal: Communications Biology

Article Title: Bmp8a is an essential positive regulator of antiviral immunity in zebrafish

doi: 10.1038/s42003-021-01811-0

Figure Lengend Snippet: Upon virus infection, the transcriptions of bmp8a are activated through the Jak-Stat1 pathway. The Bmp8a binds to BMP type I receptor Alk6a, promoting phosphorylation of Tbk1 and Irf3 to induce the expression of Ifn through p38 MAPK pathway.

Article Snippet: Antibodies from Bioss: IRF3 (1:1000, #bs-2993R), p-IRF3 (Ser386) (1:1000, #bsm-52170R), p38MAPK (1:1000, #bs-0637R), p-p38MAPK (Thr180 + Tyr182) (1:1000, #bs-2210R), Actin (1:2000, #bs-0061R).

Techniques: Infection, Expressing

Journal: Journal of Virology

Article Title: LYAR Suppresses Beta Interferon Induction by Targeting Phosphorylated Interferon Regulatory Factor 3

doi: 10.1128/JVI.00769-19

Figure Lengend Snippet: LYAR inhibits IFN-β promoter activity induced by IRF3-mediated IFN-β signaling molecules. (A to G) The effect of different doses of LYAR on the activation of IFN-β promoter induced by RIG-I (A), MAVS (B), TBK1 (C), IKKε (D), IRF3 (E), IRF3-5D (F), and IRF7 (G). HEK293T cells were transfected with IFN-β-luc, pRL-TK, plus the indicated expression plasmids of signal molecules, along with an increasing amount of HA-LYAR (0, 0.25, 0.5, and 1.0 μg). Luciferase activity was measured at 24 h posttransfection. The expression levels of LYAR and each signal molecule were detected by using Western blotting with an anti-HA antibody and an anti-Flag antibody, respectively. (H) The effect of LYAR on IRF9 induced activation of ISRE promoter. HEK293T cells were transfected with ISRE-luc, pRL-TK, HA-IRF9, and increasing amounts of Flag-LYAR (0, 0.25, 0.5, and 1.0 μg). Luciferase activity was measured at 24 h posttransfection. The expressions of LYAR and IRF9 were detected by using anti-Flag and anti-HA antibodies, respectively. The data are presented as the mean ± SD from three independent experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001; all by two-tailed Student’s t test).

Article Snippet: Antibodies used for Western blotting, immunoprecipitation, and indirect immunofluorescence were anti-Flag M2 mouse monoclonal antibody (F3165; Sigma, USA), anti-LYAR mouse polyclonal antibody (H00055646-B01P; Abnova, China), anti-HA and -GAPDH mouse monoclonal antibodies (PMK013C and PMK043F; PMK Bio, China), anti-histone 3.1 polyclonal rabbit antibody (p30266; Abmart, USA), anti-IRF3 rabbit monoclonal antibody (11904; Cell Signaling Technology, USA), anti-Ser386 phosphorylated IRF3 polyclonal rabbit antibody (AP0091; ABclonal Technology, USA), anti-CBP rabbit monoclonal antibody (D6C5; Cell Signaling Technology, USA), and Alexa Fluor 488-conjugated AffiniPure goat anti-rabbit and Alexa Fluor 594-conjugated AffiniPure goat anti-mouse secondary antibodies (SA00006-2 and SA00006-3; Proteintech, USA).

Techniques: Activity Assay, Activation Assay, Transfection, Expressing, Luciferase, Western Blot, Two Tailed Test

Journal: Journal of Virology

Article Title: LYAR Suppresses Beta Interferon Induction by Targeting Phosphorylated Interferon Regulatory Factor 3

doi: 10.1128/JVI.00769-19

Figure Lengend Snippet: LYAR interacts with phosphorylated IRF3. (A) The interaction between LYAR and IRF3 in transfected cells. HEK293T cells were cotransfected with the indicated plasmids for 24 h. Co-IP was performed using an anti-Flag (left) or anti-HA (right) antibody, followed by Western blotting to detect the LYAR and IRF3 with the anti-HA and anti-Flag antibodies, respectively. (B) The interactions between LYAR and IRF3-5D or IRF3-5D mutants (N-terminal domain, NTD; amino acids 1 to 197 and C-terminal domain, CTD; amino acids 198 to 427). HEK293T cells were transfected with the indicated plasmids for 24 h. Co-IP was conducted by using an anti-Flag (left) or anti-HA (right) mouse antibody. (C) The interaction between endogenous LYAR and IRF3. A549 cells were infected with Sev for 12 h, and Co-IP was performed using an anti-LYAR mouse antibody or mouse IgG. The endogenous LYAR and coprecipitated IRF3 were detected by using an anti-LYAR antibody and an anti-Ser386 phosphorylated IRF3 antibody, respectively. Mouse IgG served as the negative control, and GAPDH was used as a loading control. (D) Colocalization of LYAR and IRF3. HeLa cells were transfected individually with HA-LYAR or Flag-IRF3 or were cotransfected with HA-LYAR and Flag-IRF3 or Flag-IRF3-5D. Cells were fixed at 24 h posttransfection and stained for LYAR (red) and IRF3 (green) using the anti-HA rabbit antibodies and anti-Flag mouse antibodies, followed by immunostaining with the Alexa Fluor 594-conjugated AffiniPure goat anti-rabbit secondary antibodies and Alexa Fluor 488-conjugated AffiniPure goat anti-mouse antibodies. DAPI was used to stain for the nucleus (blue). The boxed region was enlarged and is shown on the right. Colocalization was quantified using the ZEN Blue software (Zeiss, Germany), and the Pearson’s correlation coefficient (Rr, values from 0.5 to 1.0 indicating colocalization) and overlap coefficient according to Manders (R, values from 0.6 to 1.0 indicating colocalization) are shown below. Images are representative of three independent experiments. Scale bar, 10 μm.

Article Snippet: Antibodies used for Western blotting, immunoprecipitation, and indirect immunofluorescence were anti-Flag M2 mouse monoclonal antibody (F3165; Sigma, USA), anti-LYAR mouse polyclonal antibody (H00055646-B01P; Abnova, China), anti-HA and -GAPDH mouse monoclonal antibodies (PMK013C and PMK043F; PMK Bio, China), anti-histone 3.1 polyclonal rabbit antibody (p30266; Abmart, USA), anti-IRF3 rabbit monoclonal antibody (11904; Cell Signaling Technology, USA), anti-Ser386 phosphorylated IRF3 polyclonal rabbit antibody (AP0091; ABclonal Technology, USA), anti-CBP rabbit monoclonal antibody (D6C5; Cell Signaling Technology, USA), and Alexa Fluor 488-conjugated AffiniPure goat anti-rabbit and Alexa Fluor 594-conjugated AffiniPure goat anti-mouse secondary antibodies (SA00006-2 and SA00006-3; Proteintech, USA).

Techniques: Transfection, Co-Immunoprecipitation Assay, Western Blot, Infection, Negative Control, Staining, Immunostaining, Software

Journal: Journal of Virology

Article Title: LYAR Suppresses Beta Interferon Induction by Targeting Phosphorylated Interferon Regulatory Factor 3

doi: 10.1128/JVI.00769-19

Figure Lengend Snippet: LYAR does not affect the phosphorylation and nuclear import of IRF3. (A and B) Effect of LYAR on IRF3 phosphorylation. HEK293T cells transfected with HA-LYAR (A) or si-LYAR (B) were infected with Sev for 12 h. The protein and phosphorylation levels of IRF3 were detected with Western blotting using an anti-IRF3 rabbit antibody and anti-Ser386 phosphorylated IRF3 rabbit antibody, respectively. GAPDH served as the loading control, and the band intensities were analyzed using the software ImageJ (NIH), and the relative p-IRF3 levels (p-IRF3/GAPDH) are shown below. (C and D) Confocal microscopy analysis of the nucleocytoplasmic distribution of IRF3 in Sev-infected LYAR overexpression cells. A549 cells transfected with HA-LYAR or HA were infected with Sev for 6 h, and endogenous IRF3 and HA-LYAR were detected by IFA using an anti-IRF3 rabbit antibody and anti-HA mouse antibody, followed by immunostaining with 594-conjugated AffiniPure goat anti-rabbit secondary antibodies (red) and Alexa Fluor 488-conjugated AffiniPure goat anti-mouse antibodies (green). Scale bar, 10 μm. Images are representative of three independent experiments. Six images in a random field of view from each sample were scored by the cell counter plugin of ImageJ (NIH). (D) The ratios of IRF3 nuclear localization cells/total cells were analyzed from three independent experiments (means ± SD from three independent experiments). (E) Western blot analysis of the distribution of IRF3 in the cytoplasmic and nuclear fractions in Sev-infected LYAR overexpression cells. A549 cells were treated as described for panel C. Cells were harvested and subjected to nuclear and cytoplasmic fractionation. Western blotting using an anti-IRF3 antibody to determine the IRF3 content of the nuclear (Nuc) and cytoplasmic (Cyt) fractions, respectively. Histone 3.1 was used as a nuclear loading control and marker, and GAPDH, as a cytosolic loading control and marker. The band intensities were quantified using ImageJ, and the relative IRF3 levels (IRF3/GAPDH or histone 3.1) are shown below the images.

Article Snippet: Antibodies used for Western blotting, immunoprecipitation, and indirect immunofluorescence were anti-Flag M2 mouse monoclonal antibody (F3165; Sigma, USA), anti-LYAR mouse polyclonal antibody (H00055646-B01P; Abnova, China), anti-HA and -GAPDH mouse monoclonal antibodies (PMK013C and PMK043F; PMK Bio, China), anti-histone 3.1 polyclonal rabbit antibody (p30266; Abmart, USA), anti-IRF3 rabbit monoclonal antibody (11904; Cell Signaling Technology, USA), anti-Ser386 phosphorylated IRF3 polyclonal rabbit antibody (AP0091; ABclonal Technology, USA), anti-CBP rabbit monoclonal antibody (D6C5; Cell Signaling Technology, USA), and Alexa Fluor 488-conjugated AffiniPure goat anti-rabbit and Alexa Fluor 594-conjugated AffiniPure goat anti-mouse secondary antibodies (SA00006-2 and SA00006-3; Proteintech, USA).

Techniques: Transfection, Infection, Western Blot, Software, Confocal Microscopy, Over Expression, Immunostaining, Fractionation, Marker

Journal: Journal of Virology

Article Title: LYAR Suppresses Beta Interferon Induction by Targeting Phosphorylated Interferon Regulatory Factor 3

doi: 10.1128/JVI.00769-19

Figure Lengend Snippet: LYAR impedes IRF3 binding onto ISRE. (A) The effect of LYAR on IRF3 binding onto ISRE. HEK293T cells cotransfected with Flag-IRF3 and HA-LYAR or HA were infected with Sev for 12 h. Biotinylated and unbiotinylated ISRE were mixed with the cell lysates at the indicated ratios and incubated at 4°C for 4 h. The beads were then added and incubated for 2 h at 4°C, followed by washing four times with PBS. Western blotting was done to detect IRF3 and LYAR by using an anti-Flag and anti-HA antibody, respectively. The Flag-IRF3 levels are shown below. (B) The effect of LYAR on the interaction between IRF3 and CBP/p300. HEK293T cells cotransfected with Flag-IRF3 and HA-LYAR or HA were infected with Sev for 12 h. Co-IP assay was performed using an anti-Flag antibody to immunoprecipitate endogenous CBP. The relative precipitated CBP/Flag-IRF3 ratios are shown below.

Article Snippet: Antibodies used for Western blotting, immunoprecipitation, and indirect immunofluorescence were anti-Flag M2 mouse monoclonal antibody (F3165; Sigma, USA), anti-LYAR mouse polyclonal antibody (H00055646-B01P; Abnova, China), anti-HA and -GAPDH mouse monoclonal antibodies (PMK013C and PMK043F; PMK Bio, China), anti-histone 3.1 polyclonal rabbit antibody (p30266; Abmart, USA), anti-IRF3 rabbit monoclonal antibody (11904; Cell Signaling Technology, USA), anti-Ser386 phosphorylated IRF3 polyclonal rabbit antibody (AP0091; ABclonal Technology, USA), anti-CBP rabbit monoclonal antibody (D6C5; Cell Signaling Technology, USA), and Alexa Fluor 488-conjugated AffiniPure goat anti-rabbit and Alexa Fluor 594-conjugated AffiniPure goat anti-mouse secondary antibodies (SA00006-2 and SA00006-3; Proteintech, USA).

Techniques: Binding Assay, Infection, Incubation, Western Blot, Co-Immunoprecipitation Assay

Journal: Journal of Virology

Article Title: LYAR Suppresses Beta Interferon Induction by Targeting Phosphorylated Interferon Regulatory Factor 3

doi: 10.1128/JVI.00769-19

Figure Lengend Snippet: Proposed model for the role of LYAR in the antiviral innate immune responses. Virus-induced IFN-β increases LYAR expression (a and b). In turn, LYAR suppresses IFN-β and ISG production by impeding the DNA binding ability of IRF3, ultimately resulting in enhanced virus replication (c to e).

Article Snippet: Antibodies used for Western blotting, immunoprecipitation, and indirect immunofluorescence were anti-Flag M2 mouse monoclonal antibody (F3165; Sigma, USA), anti-LYAR mouse polyclonal antibody (H00055646-B01P; Abnova, China), anti-HA and -GAPDH mouse monoclonal antibodies (PMK013C and PMK043F; PMK Bio, China), anti-histone 3.1 polyclonal rabbit antibody (p30266; Abmart, USA), anti-IRF3 rabbit monoclonal antibody (11904; Cell Signaling Technology, USA), anti-Ser386 phosphorylated IRF3 polyclonal rabbit antibody (AP0091; ABclonal Technology, USA), anti-CBP rabbit monoclonal antibody (D6C5; Cell Signaling Technology, USA), and Alexa Fluor 488-conjugated AffiniPure goat anti-rabbit and Alexa Fluor 594-conjugated AffiniPure goat anti-mouse secondary antibodies (SA00006-2 and SA00006-3; Proteintech, USA).

Techniques: Expressing, Binding Assay