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Bioss p irf3
P Irf3, supplied by Bioss, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/irf3+(ser386)+polyclonal+antibody/IRF3+(Ser386)+Polyclonal+Antibody/ppr0934931-86-11-13
Average 93 stars, based on 4 article reviews

p irf3 - by Bioz Stars, 2026-10

93/100 stars

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Article Title: O-GlcNAcylation of irf3 by a novel Zebrafish O-GlcNAc transferase isoform Ogtb_tv3 facilitates its sufficient activation and innate antiviral response
Article Snippet: .. Proteins were extracted and immunoblotted using antibodies against IRF3 (bs-2993R; Bioss), p-IRF3 (bs-9278R; Bioss), β-O-Linked N-Acetylglucosamine, GAPDH (60004-1-Ig; Proteintech), βActin (sc-81178; Santa Cruz), and FLAG tag. .. The impact of DON and OSMI-1 on IRF3 activation was evaluated by treating EPC cells overnight with 20 μM DON (D214; Sigma) or 20 μM OSMI-1 (HY-119738; MCE), followed by SVCV infection at an MOI of 1 for 12 hours.

other:

Article Title: sNASP Mutation Aggravates to the TLR4-Mediated Inflammation in SLE by TAK1 Pathway
Article Snippet: Anti-p-IRF3 (catalog number: bs-9278R) was from Bioss Technology.

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PHB1 negatively regulates the IFN-β signal pathway by targeting IRF3 signaling. (A–J) The effects of (A–E) PHB1 overexpression or (F-J) PHB1 silencing on the activity of IFN-β promoter induced by adaptor molecules including (A and E) RIG-I, (B and G) MAVS, (C and H) IKK-ε, (D and I) TBK1, and (E and J) IRF3 were examined. HEK293T cells were transfected with IFN-β-luc, pRL-TK, and the indicated plasmids expressing the signal molecules, along with Flag-PHB1 or si-PHB1. Luciferase activities were measured at 24 hpi. The expression levels of PHB1 and each signal molecule were detected by western blot. The data are presented as the mean ± SD from three independent experiments. (K–M) PHB1 co-purified with IRF3. (K and L) The co-purification between PHB1 and IRF3 in transfected cells. HEK293T cells were co-transfected with the indicated plasmids for 24 h. Co-IP was performed using either (K) anti-Flag or (L) anti-HA antibodies, followed by western blot. (M) The co-purification between endogenous PHB1 and IRF3. Co-IP was performed using an anti-IRF3 rabbit antibody or rabbit IgG with or without SeV infection. The endogenous IRF3 and coprecipitated PHB1 were detected using an anti-IRF3 antibody and an anti-PHB1 antibody, respectively. Rabbit IgG was used as the negative control. GAPDH was used as a control. The band intensities were quantified by ImageJ, and the relative levels of PHB1/IRF3 are shown (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Journal: Journal of Virology

Article Title: Prohibitin1 facilitates viral replication by impairing the RIG-I-like receptor signaling pathway

doi: 10.1128/jvi.00926-23

Figure Lengend Snippet: PHB1 negatively regulates the IFN-β signal pathway by targeting IRF3 signaling. (A–J) The effects of (A–E) PHB1 overexpression or (F-J) PHB1 silencing on the activity of IFN-β promoter induced by adaptor molecules including (A and E) RIG-I, (B and G) MAVS, (C and H) IKK-ε, (D and I) TBK1, and (E and J) IRF3 were examined. HEK293T cells were transfected with IFN-β-luc, pRL-TK, and the indicated plasmids expressing the signal molecules, along with Flag-PHB1 or si-PHB1. Luciferase activities were measured at 24 hpi. The expression levels of PHB1 and each signal molecule were detected by western blot. The data are presented as the mean ± SD from three independent experiments. (K–M) PHB1 co-purified with IRF3. (K and L) The co-purification between PHB1 and IRF3 in transfected cells. HEK293T cells were co-transfected with the indicated plasmids for 24 h. Co-IP was performed using either (K) anti-Flag or (L) anti-HA antibodies, followed by western blot. (M) The co-purification between endogenous PHB1 and IRF3. Co-IP was performed using an anti-IRF3 rabbit antibody or rabbit IgG with or without SeV infection. The endogenous IRF3 and coprecipitated PHB1 were detected using an anti-IRF3 antibody and an anti-PHB1 antibody, respectively. Rabbit IgG was used as the negative control. GAPDH was used as a control. The band intensities were quantified by ImageJ, and the relative levels of PHB1/IRF3 are shown (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Article Snippet: Antibodies used for western blot, immunoprecipitation, and indirect immunofluorescence assays included the following: anti-Flag mouse monoclonal antibody (F3165; Sigma, St. Louis, MO, USA), anti-HA and -GAPDH mouse monoclonal antibodies (PMK013C and PMK043F; PMK Bio, Wuhan, China), anti-NP rabbit polyclonal antibody (GTX125989; GeneTeX, Irvine, CA, USA), anti-IRF3 rabbit monoclonal antibody (11904; CST, Boston, MA, USA), anti-GFP rabbit monoclonal and anti-Ser386 phosphorylated IRF3 rabbit polyclonal antibodies (AE078 and AP0091; ABclonal, Wuhan, China), anti-PHB1 rabbit polyclonal antibody (ab28172; Abcam, Boston, MA, USA), anti-PHB1 mouse monoclonal antibody (60092-1-Ig; Proteintech, Wuhan, China), anti-TOMM20 rabbit polyclonal antibody (11802-1-AP; Proteintech), anti-LaminA/C rabbit polyclonal antibody (10298-1-AP; Proteintech), HRP goat anti-rabbit IgG (H + L) and HRP goat anti-mouse IgG (H + L) (AS014 and AS003; ABclonal), HRP goat anti-rabbit IgG HCS and HRP goat anti-mouse IgG HCS (A25222 and A25112; Abbkine, Wuhan, China), HRP mouse anti-rabbit IgG LCS and HRP goat anti-mouse IgG LCS (A25022 and A25012; Abbkine), 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).

Techniques: Over Expression, Activity Assay, Transfection, Expressing, Luciferase, Western Blot, Purification, Copurification, Co-Immunoprecipitation Assay, Infection, Negative Control, Control

TBK1-IRF3 complex formation, IRF3 dimer formation, and IRF3 phosphorylation were not affected by PHB1. (A) The effects of PHB1 on the formation of the TBK1-IRF3 complex. HEK293T cells were co-transfected with the indicated plasmids for 24 h. Co-IP was performed using an anti-HA antibody, followed by western blotting to detect the expression of the plasmids and coprecipitated proteins. (B and C) The effect of PHB1 on IRF3 phosphorylation. HEK293T cells transfected with (B) Flag-PHB1 or (C) si-PHB1 were infected with SeV for 12 h. The protein and phosphorylation levels of IRF3 were detected by western blot using an anti-IRF3 antibody and an anti-Ser386 phosphorylated IRF3 antibody, respectively. GAPDH served as a control. (D and E) The effects of PHB1 on the formation of IRF3 dimers. HEK293T cells were co-transfected with the indicated plasmids for 24 h. Co-IP was performed using an anti-HA antibody, followed by western blotting to detect the expression of the plasmids and coprecipitated proteins. (F) The effects of endogenous PHB1 knockdown on TBK1-IRF3 and IRF3 dimer formation. HEK293T cells were treated with si-PHB1 or si-NC for 24 h. Co-IP was performed using an anti-IRF3 antibody, followed by western blotting to detect the expression of coprecipitated proteins. GAPDH was used as a control. The relative band intensities were quantified by ImageJ.

Journal: Journal of Virology

Article Title: Prohibitin1 facilitates viral replication by impairing the RIG-I-like receptor signaling pathway

doi: 10.1128/jvi.00926-23

Figure Lengend Snippet: TBK1-IRF3 complex formation, IRF3 dimer formation, and IRF3 phosphorylation were not affected by PHB1. (A) The effects of PHB1 on the formation of the TBK1-IRF3 complex. HEK293T cells were co-transfected with the indicated plasmids for 24 h. Co-IP was performed using an anti-HA antibody, followed by western blotting to detect the expression of the plasmids and coprecipitated proteins. (B and C) The effect of PHB1 on IRF3 phosphorylation. HEK293T cells transfected with (B) Flag-PHB1 or (C) si-PHB1 were infected with SeV for 12 h. The protein and phosphorylation levels of IRF3 were detected by western blot using an anti-IRF3 antibody and an anti-Ser386 phosphorylated IRF3 antibody, respectively. GAPDH served as a control. (D and E) The effects of PHB1 on the formation of IRF3 dimers. HEK293T cells were co-transfected with the indicated plasmids for 24 h. Co-IP was performed using an anti-HA antibody, followed by western blotting to detect the expression of the plasmids and coprecipitated proteins. (F) The effects of endogenous PHB1 knockdown on TBK1-IRF3 and IRF3 dimer formation. HEK293T cells were treated with si-PHB1 or si-NC for 24 h. Co-IP was performed using an anti-IRF3 antibody, followed by western blotting to detect the expression of coprecipitated proteins. GAPDH was used as a control. The relative band intensities were quantified by ImageJ.

Techniques: Phospho-proteomics, Transfection, Co-Immunoprecipitation Assay, Western Blot, Expressing, Infection, Control, Knockdown

$PHB1 inhibits the nuclear import of IRF3. The effects of PHB1 (A–C) overexpression or (D–F) knockdown on IRF3 nucleus import. (A, D) Western blot analysis was performed to examine the distribution of IRF3 in cytoplasmic and nuclear fractions in PHB1 (A) overexpression or (D) knockdown cells, with or without SeV infection. Cells were harvested and subjected to nuclear fractionation (Nuc) and cytoplasmic fractionation (Cyt). An anti-IRF3 antibody was used to determine the content of IRF3 in the nuclear and cytoplasmic fractions by western blot. GAPDH and Lamin A/C were used as cytosolic and nuclear controls, respectively. The band intensities were quantified using ImageJ, and the relative levels of IRF3 (IRF3/GAPDH or Lamin A/C) are shown below the images. (B, E) Confocal microscopy analysis of the nucleocytoplasmic distribution of IRF3 in PHB1 (B) overexpression or (E) knockdown cells, with or without SeV infection. (B) A549 cells were transfected with HA-IRF3 and Flag-PHB1 or Flag vectors and infected with SeV for 6 h or not (mock group). HA-IRF3 and Flag-PHB1 were detected by IFA using an anti-HA rabbit antibody and an anti-Flag mouse antibody. (E) A549 cells were transfected with si-PHB1 or si-NC and infected with SeV for 6 h or not (mock group). IRF3 and PHB1 were detected by IFA using an anti-IRF3 rabbit antibody and an anti-PHB1 mouse antibody, followed by immunostaining with 594-conjugated AffiniPure goat anti-rabbit secondary antibodies (red) and Alexa Fluor 488-conjugated AffiniPure goat anti-mouse antibodies (green), respectively. Scale bar = 20 µm. (C, F) The relative nucleus IRF3 distributions in (B, E) were analyzed by ImageJ and subjected to statistical analysis, respectively. (****, P < 0.0001).$

Journal: Journal of Virology

Article Title: Prohibitin1 facilitates viral replication by impairing the RIG-I-like receptor signaling pathway

doi: 10.1128/jvi.00926-23

Figure Lengend Snippet: PHB1 inhibits the nuclear import of IRF3. The effects of PHB1 (A–C) overexpression or (D–F) knockdown on IRF3 nucleus import. (A, D) Western blot analysis was performed to examine the distribution of IRF3 in cytoplasmic and nuclear fractions in PHB1 (A) overexpression or (D) knockdown cells, with or without SeV infection. Cells were harvested and subjected to nuclear fractionation (Nuc) and cytoplasmic fractionation (Cyt). An anti-IRF3 antibody was used to determine the content of IRF3 in the nuclear and cytoplasmic fractions by western blot. GAPDH and Lamin A/C were used as cytosolic and nuclear controls, respectively. The band intensities were quantified using ImageJ, and the relative levels of IRF3 (IRF3/GAPDH or Lamin A/C) are shown below the images. (B, E) Confocal microscopy analysis of the nucleocytoplasmic distribution of IRF3 in PHB1 (B) overexpression or (E) knockdown cells, with or without SeV infection. (B) A549 cells were transfected with HA-IRF3 and Flag-PHB1 or Flag vectors and infected with SeV for 6 h or not (mock group). HA-IRF3 and Flag-PHB1 were detected by IFA using an anti-HA rabbit antibody and an anti-Flag mouse antibody. (E) A549 cells were transfected with si-PHB1 or si-NC and infected with SeV for 6 h or not (mock group). IRF3 and PHB1 were detected by IFA using an anti-IRF3 rabbit antibody and an anti-PHB1 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), respectively. Scale bar = 20 µm. (C, F) The relative nucleus IRF3 distributions in (B, E) were analyzed by ImageJ and subjected to statistical analysis, respectively. (****, P < 0.0001).

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

PHB1 disrupts the formation of IRF3-importin α1 and IRF3-importin α5 complexes. (A) The complex formation between IRF3 and importin α in transfected cells. HEK293T cells were co-transfected with Flag-IRF3 and HA-importin α1/α3/α5/α7 for 24 h. Co-IPs were performed using an anti-Flag antibody. (B and C) The effects of PHB1 on the complex formation between exogenous IRF3 and importin α1 or importin α5. HEK293T cells were co-transfected with Flag-IRF3 and (B) HA-importin α1 or (C) HA-importin α5, along with increasing amounts of Flag-PHB1 (0, 0.3, and 1.0 µg) for 24 h. Co-IPs were performed using an anti-HA antibody, followed by western blotting to detect the expression of plasmids and coprecipitated proteins. The band intensities were quantified by ImageJ, and the relative levels of IRF3/importins are shown. (D) The effects of PHB1 on the complex formation between endogenous IRF3 and importin α1 or importin α5. HEK293T cells were treated with si-PHB1 or si-NC for 24 h, and co-IPs were performed using an anti-IRF3 antibody, followed by western blot to detect the expression of plasmids and coprecipitated proteins. The band intensities were quantified by ImageJ, and the relative levels of IRF3/importins are shown. (E) The co-purification between importin α1 and importin α5 with GFP-IRF3-NLS. HEK293T cells were co-transfected with the indicated plasmids for 24 h. Co-IP was performed using an anti-HA antibody, followed by western blot to detect importin α1 or importin α5 and GFP-IRF3-NLS with the anti-HA and anti-GFP antibodies, respectively. (F) The co-purification between PHB1 and IRF3 or its truncations in transfected cells. HEK293T cells were co-transfected with the indicated plasmids for 24 h. Co-IP was performed using an anti-GFP antibody, followed by western blot to detect PHB1 with the anti-Flag and detect IRF3 or its truncations with anti-GFP antibodies. GAPDH was used as a control.

Journal: Journal of Virology

Article Title: Prohibitin1 facilitates viral replication by impairing the RIG-I-like receptor signaling pathway

doi: 10.1128/jvi.00926-23

Figure Lengend Snippet: PHB1 disrupts the formation of IRF3-importin α1 and IRF3-importin α5 complexes. (A) The complex formation between IRF3 and importin α in transfected cells. HEK293T cells were co-transfected with Flag-IRF3 and HA-importin α1/α3/α5/α7 for 24 h. Co-IPs were performed using an anti-Flag antibody. (B and C) The effects of PHB1 on the complex formation between exogenous IRF3 and importin α1 or importin α5. HEK293T cells were co-transfected with Flag-IRF3 and (B) HA-importin α1 or (C) HA-importin α5, along with increasing amounts of Flag-PHB1 (0, 0.3, and 1.0 µg) for 24 h. Co-IPs were performed using an anti-HA antibody, followed by western blotting to detect the expression of plasmids and coprecipitated proteins. The band intensities were quantified by ImageJ, and the relative levels of IRF3/importins are shown. (D) The effects of PHB1 on the complex formation between endogenous IRF3 and importin α1 or importin α5. HEK293T cells were treated with si-PHB1 or si-NC for 24 h, and co-IPs were performed using an anti-IRF3 antibody, followed by western blot to detect the expression of plasmids and coprecipitated proteins. The band intensities were quantified by ImageJ, and the relative levels of IRF3/importins are shown. (E) The co-purification between importin α1 and importin α5 with GFP-IRF3-NLS. HEK293T cells were co-transfected with the indicated plasmids for 24 h. Co-IP was performed using an anti-HA antibody, followed by western blot to detect importin α1 or importin α5 and GFP-IRF3-NLS with the anti-HA and anti-GFP antibodies, respectively. (F) The co-purification between PHB1 and IRF3 or its truncations in transfected cells. HEK293T cells were co-transfected with the indicated plasmids for 24 h. Co-IP was performed using an anti-GFP antibody, followed by western blot to detect PHB1 with the anti-Flag and detect IRF3 or its truncations with anti-GFP antibodies. GAPDH was used as a control.

Techniques: Transfection, Western Blot, Expressing, Copurification, Co-Immunoprecipitation Assay, Control

The PHB domain of PHB1 plays a dominant role in inhibiting IFN-β production. (A) The schematic of full-length PHB1 and PHB1 mutants. (B) Effects of PHB1 mutants on IFN-β promoter activity. HEK293T cells were transfected with pRL-TK, IFN-β-luc, PHB1 mutants, or the Flag vector. After 24 hpi, the cells were infected with SeV or not, and the luciferase report assay was used to measure the luciferase activities. (C) Effects of PHB1 mutants on the virus titers of the PR8 H1N1 virus. A549 cells transfected with PHB1 or PHB1 mutants were infected with the PR8 H1N1 virus at an MOI of 0.01. Cell supernatants were collected at the indicated time points. Virus titers were determined by TCID50 on MDCK cells (means ± SD from three independent experiments). (D and E) The co-purification between PHB1 mutants and IRF3. HEK293T cells were co-transfected with Flag-PHB1 or PHB1 mutants (D) with HA-IRF3 or (E) not for 24 h. Co-IP was performed using an anti-Flag antibody, followed by western blot to detect PHB1 or PHB1 mutants and IRF3 using anti-Flag, anti-HA, or anti-IRF3 antibodies, respectively. (ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Journal: Journal of Virology

Article Title: Prohibitin1 facilitates viral replication by impairing the RIG-I-like receptor signaling pathway

doi: 10.1128/jvi.00926-23

Figure Lengend Snippet: The PHB domain of PHB1 plays a dominant role in inhibiting IFN-β production. (A) The schematic of full-length PHB1 and PHB1 mutants. (B) Effects of PHB1 mutants on IFN-β promoter activity. HEK293T cells were transfected with pRL-TK, IFN-β-luc, PHB1 mutants, or the Flag vector. After 24 hpi, the cells were infected with SeV or not, and the luciferase report assay was used to measure the luciferase activities. (C) Effects of PHB1 mutants on the virus titers of the PR8 H1N1 virus. A549 cells transfected with PHB1 or PHB1 mutants were infected with the PR8 H1N1 virus at an MOI of 0.01. Cell supernatants were collected at the indicated time points. Virus titers were determined by TCID50 on MDCK cells (means ± SD from three independent experiments). (D and E) The co-purification between PHB1 mutants and IRF3. HEK293T cells were co-transfected with Flag-PHB1 or PHB1 mutants (D) with HA-IRF3 or (E) not for 24 h. Co-IP was performed using an anti-Flag antibody, followed by western blot to detect PHB1 or PHB1 mutants and IRF3 using anti-Flag, anti-HA, or anti-IRF3 antibodies, respectively. (ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Infection, Luciferase, Virus, Copurification, Co-Immunoprecipitation Assay, Western Blot

Proposed model for the role of PHB1 in the IFN-β signal pathway. RNA viral infections induce IFN-β production and subsequently increase the mRNA and protein levels of PHB1. In return, PHB1 restrains the IFN-β signal pathway by inhibiting the nucleus import of IRF3, ultimately facilitating virus replication.

Journal: Journal of Virology

Article Title: Prohibitin1 facilitates viral replication by impairing the RIG-I-like receptor signaling pathway

doi: 10.1128/jvi.00926-23

Figure Lengend Snippet: Proposed model for the role of PHB1 in the IFN-β signal pathway. RNA viral infections induce IFN-β production and subsequently increase the mRNA and protein levels of PHB1. In return, PHB1 restrains the IFN-β signal pathway by inhibiting the nucleus import of IRF3, ultimately facilitating virus replication.

Techniques: Virus

Journal: Journal of Virology

Article Title: Prohibitin1 facilitates viral replication by impairing the RIG-I-like receptor signaling pathway

doi: 10.1128/jvi.00926-23

Figure Lengend Snippet: Primers used for PCR

Techniques:

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).

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

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).

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).

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

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.

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.

Techniques: Infection, Expressing

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