Journal: bioRxiv

Article Title: Ring-finger protein 34 facilitates nervous necrosis virus evading antiviral innate immunity by targeting TBK1 and IRF3 for ubiquitination and degradation

doi: 10.1101/2022.12.05.519093

Figure Lengend Snippet: (A-D) HEK 293T cells were transfected with pCMV-Myc-RNF34 and pCMV-Flag-MAVS, pCMV-Flag-TRAF3, pCMV-Flag-TBK1 or pCMV-Flag-IRF3 plasmids, respectively. At 24 h post transfection, the cell lysates were subjected to co-immunoprecipitation analysis with anti-Myc magnetic beads as indicated. (E-F) pCMV-Myc-RNF34 and pCMV-Flag-TBK1 or pCMV-Flag-IRF3 plasmids were transfected into HEK 293T cells for immunofluorescence analysis by using anti-Myc (red) and anti-Flag (green) antibodies. Nuclei were stained with DAPI. (G-H) Plasmids of RNF34 from zebrafish (zbRNF34) and marine medaka (mmRNF34) were transfected into HEK 293T cells, together with pCMV-Myc-TBK1 or pCMV-Myc-IRF3 plasmids, respectively. At 24 h post transfection, the cell lysates were subjected to co-immunoprecipitation analysis with anti-Flag magnetic beads as indicated. (I-J) HEK 293T cells were transfected with Flag-Tagged RNF34 mutant plasmids (RNF34ΔRING and RNF34ΔZinc) as indicated for 24 h, the cell lysates were subjected to co-immunoprecipitation analysis with anti-Flag magnetic beads as above.

Article Snippet: Antibodies to TBK1 (bs-7497R) and IRF3 (bs-1185R) were obtained from Bioss (Beijing, China).

Techniques: Transfection, Immunoprecipitation, Magnetic Beads, Immunofluorescence, Staining, Mutagenesis

Journal: bioRxiv

Article Title: Ring-finger protein 34 facilitates nervous necrosis virus evading antiviral innate immunity by targeting TBK1 and IRF3 for ubiquitination and degradation

doi: 10.1101/2022.12.05.519093

Figure Lengend Snippet: (A-B) FHM cells were co-transfected with an increasing amount of pCMV-Myc-RNF34 (0, 100, 200, and 300 ng), pGL3-IFNh-pro-Luc and pRL-TK , together with pCMV-Flag-TBK1 (A) or pCMV-Flag-IRF3 (B) plasmids, respectively, for luciferase activity analysis. (C-D) RNF34 mutant plasmids were transfected into FHM cells for 24 h, along with pCMV-Myc-TBK1 (C) or pCMV-Myc-IRF3 (D) plasmids, respectively. Reporter assays were performed as above (n = 3). * p < 0.05; ** p < 0.01.

Article Snippet: Antibodies to TBK1 (bs-7497R) and IRF3 (bs-1185R) were obtained from Bioss (Beijing, China).

Techniques: Transfection, Luciferase, Activity Assay, Mutagenesis

Journal: bioRxiv

Article Title: Ring-finger protein 34 facilitates nervous necrosis virus evading antiviral innate immunity by targeting TBK1 and IRF3 for ubiquitination and degradation

doi: 10.1101/2022.12.05.519093

Figure Lengend Snippet: (A-B) qRT-PCR analysis of RNF34, TBK1 and IRF3 mRNA expression in LJB cells with pCMV-Myc-RNF34 overexpressed (A) or RNF34-knock down (B), following infection with RGNNV for 24 h. (C-D) HEK 293T cells were transfected with the empty vector or pCMV-Myc-RNF34 plasmid (0, 0.5, and 1 μg), together with pCMV-Flag-TBK1 (C) or pCMV-Flag-IRF3 (D) plasmids, respectively. At 24 h post transfection, the cells were lysed for immunoblot assays with indicated antibodies. (E-F) LJB cells transfected with pCMV-Myc-RNF34 (0, 1.5, and 3 μg) without (E) or with RGNNV infection (F) were subjected to immunoblot assays using anti-TBK1 and anti-IRF3 antibodies.

Article Snippet: Antibodies to TBK1 (bs-7497R) and IRF3 (bs-1185R) were obtained from Bioss (Beijing, China).

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

Journal: bioRxiv

Article Title: Ring-finger protein 34 facilitates nervous necrosis virus evading antiviral innate immunity by targeting TBK1 and IRF3 for ubiquitination and degradation

doi: 10.1101/2022.12.05.519093

Figure Lengend Snippet: (A-B) HEK 293T cells were transfected with pCMV-Myc-RNF34 plasmid, along with the pCMV-Flag-TBK1 (A) or pCMV-Flag-IRF3 (B) plasmids, and then stimulated with increasing amount of MG132 (10 and 20 μM) for 6 h. The cells were lysed for immunoblot assays with indicated antibodies. (C-D) HEK 293T cells were cotransfected with pCMV-Myc-RNF34, HA-K27, HA-K48 , or HA-K63 , along with the pCMV-Flag-TBK1 (C) or pCMV-Flag-IRF3 (D) plasmids for 24 h. Afterwards, the cells were lysed for co-immunoprecipitation analysis with anti-Flag antibodies as indicated. (E-F) Luciferase activity of IFNh promoter in FHM cells transfected with pCMV-Myc-RNF34, HA-K27, HA-K48 , or HA-K63 , along with the pCMV-Flag-TBK1 (E) or pCMV-Flag-IRF3 (F) plasmids for 24 h. Data is collected from three independent experiments and presented as mean ± SD. * p < 0.05; ** p < 0.01.

Article Snippet: Antibodies to TBK1 (bs-7497R) and IRF3 (bs-1185R) were obtained from Bioss (Beijing, China).

Techniques: Transfection, Plasmid Preparation, Western Blot, Immunoprecipitation, Luciferase, Activity Assay

Journal: bioRxiv

Article Title: Ring-finger protein 34 facilitates nervous necrosis virus evading antiviral innate immunity by targeting TBK1 and IRF3 for ubiquitination and degradation

doi: 10.1101/2022.12.05.519093

Figure Lengend Snippet: (A) pCMV-Myc-RNF34 and pCMV-Flag-TBK1 or pEGFP-IRF3 plasmids were transfected into HEK 293T cells as indicated for immunofluorescence analysis by using anti-Myc (red) and anti-Flag (purple) antibodies. Nuclei were stained with DAPI. (B) HEK 293T cells were transfected with pEGFP-IRF3 plasmid, along with pCMV-Flag-TBK1 or pCMV-Myc-RNF34 plasmids, then the cells were lysed for the cytoplasmic proteins, nuclear proteins and total proteins extraction and subjected to immunoblot assays using anti-GFP, anti-Flag, anti-Myc, anti-Lamin B1 and anti-Actin antibodies.

Article Snippet: Antibodies to TBK1 (bs-7497R) and IRF3 (bs-1185R) were obtained from Bioss (Beijing, China).

Techniques: Transfection, Immunofluorescence, Staining, Plasmid Preparation, Western Blot

Journal: bioRxiv

Article Title: Ring-finger protein 34 facilitates nervous necrosis virus evading antiviral innate immunity by targeting TBK1 and IRF3 for ubiquitination and degradation

doi: 10.1101/2022.12.05.519093

Figure Lengend Snippet: CP interacted with RNF34 and utilized RNF34 to promote K27- and K48-linked ubiquitination and degradation of TBK1 and IRF3, thus impeding the translocation of IRF3 into the nucleus, finally suppressing the production of IFN. Schematic figure was drawn by Figdraw.

Article Snippet: Antibodies to TBK1 (bs-7497R) and IRF3 (bs-1185R) were obtained from Bioss (Beijing, China).

Techniques: Translocation Assay

Journal: Frontiers in Pharmacology

Article Title: Targeting F-Box Protein Fbxo3 Attenuates Lung Injury Induced by Ischemia-Reperfusion in Rats

doi: 10.3389/fphar.2019.00583

Figure Lengend Snippet: Effect of BC-1215 on Fbxl2, Fbxo3, and TRAF1–6 protein expressions in lung tissue. Western blot and densitometry analysis of TRAF1 (A) , TRAF2 (B) , TRAF3 (C) , TRAF4 (D) , TRAF5 (E) , TRAF6 (F) , Fbxo3 (G) , and Fbxl2 (H) protein in the lung tissue. β-actin served as loading control for cytoplasmic proteins. Representative blots are shown. Ischemia-reperfusion (I/R) significantly increased protein expression of TRAF1–6 and Fbxo3 and decreased Fbxl2 protein expression in lung tissue compared with the control group. BC-1215 significantly decreased the degree of TRAF1-6 and Fbxo3 protein expression and increased Fbxl2 protein expression in the I/R group. Data are expressed as mean ± SD ( n = 3 per group). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the control group; + p < 0.05, ++ p < 0.01, +++ p < 0.001 compared with the I/R group.

Article Snippet: The blots were probed with primary antibodies against TRAF1 (catalog number: RG2234242C), TRAF2 (catalog number: RG2234515), Fbxo3 (catalog number: RF22253318), Fbxl2 (catalog number: RF2225661H; 1:500, Thermo Fisher Scientific, Rockford, IL, USA), TRAF3 (catalog number: TA322871), and TRAF5 (catalog number: TA323403; 1:500, OriGene Technologies, Rockville, MD, USA), TRAF4 (catalog number: OAAF07255-10; 1:500, Avivasysbio, San Diego, CA, USA), TRAF6 (catalog number: SC-8409; 1:500, Santa Cruz Biotechnology, Dallas, TX, USA), B-cell lymphoma (Bcl)-2 (catalog number: SC-7382; 1:200, Santa Cruz Biotechnology, Dallas, TX, USA), NF-κB p65 (catalog number: 8242), phospho-NF-κB p65 (catalog number: 3033), inhibitor of NF-κB (IκB)-α (catalog number: 4812), extracellular signal-related protein kinase 1/2 (ERK1/2) (catalog number: 9102), phosho-ERK1/2 (catalog number: 9106), c-Jun N-terminal kinase (JNK; catalog number: 9252), phospho-JNK (catalog number: 9251), p38 protein kinase (p38; catalog number: 9228), phospho-p38 (catalog number: 4511) and cleaved caspase-3 (catalog number: 9664), proliferating cell nuclear antigen (PCNA) (catalog number: 2586; 1:1000, Cell Signaling Technology, Danvers, MA, USA), and β-actin (catalog number: A5441; 1:10000, Sigma Chemical Company, St. Louis, MO, USA).

Techniques: Western Blot, Expressing