Journal: Neuroimmunomodulation

Article Title: MiR-124 Reduced Neuroinflammation after Traumatic Brain Injury by Inhibiting TRAF6.

doi: 10.1159/000528502

Figure Lengend Snippet: Fig. 5. Upregulation of miR-124 inhibited the activation of TLR4 pathway after TBI. a–e TBI increased the expression of TLR4 and its downstream molecules MyD88/TRAF6/IRAK1/NF-κB p65. MiR-124 mimics reduced the expression of these proteins, but miR-124 inhibitors produced the contrary effects. n = 12 in sham group, n = 12 in other three groups. p p < 0.05 versus sham group, #p < 0.05 versus TBI group.

Article Snippet: Equivalent amount of protein (40 μg) was loaded and separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane at 4°C for 50 min. Membranes were blocked with 5% nonfat milk solution in Tris-buffered saline with 0.1% Triton X-100 (TBST) for 1 h and then incubated overnight at 4°C with appropriate primary antibodies as follows: rabbit anti-mouse TLR4 antibody (Thermo Fisher, USA), rabbit anti-mouse myeloid differentiation factor 88 (MyD88) antibody (Santa Cruz, USA), rabbit anti-mouse TNFRassociated factor (TRAF6) antibody (Novus Biologicals, USA), rabbit anti-mouse NF-κB p65 antibody (GeneTex, USA), and rabbit anti-β-actin antibody (Proteintech, USA).

Techniques: Activation Assay, Expressing, Produced

Journal: Neuroimmunomodulation

Article Title: MiR-124 Reduced Neuroinflammation after Traumatic Brain Injury by Inhibiting TRAF6.

doi: 10.1159/000528502

Figure Lengend Snippet: Fig. 6. MiR-124 targeted TRAF6. a, b LPS elevated the expression of TRAF6 in microglial cells, upregulation of miR-124 declined the expression of TRAF6, while downregulation of miR-124 elevated the expression of TRAF6. c MiR-124 and TRAF6 had the latent binding sites. d The dual-luciferase reporter activity examina- tions of the targeting of miR-124 with TRAF6. e RNA-immunoprecipitation test of the enrichment of miR-124 with TRAF6 in Ago2 magnetic beads. n = 6 in each group, p p < 0.05 versus control group, #p < 0.05 versus LPS group.

Article Snippet: Equivalent amount of protein (40 μg) was loaded and separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane at 4°C for 50 min. Membranes were blocked with 5% nonfat milk solution in Tris-buffered saline with 0.1% Triton X-100 (TBST) for 1 h and then incubated overnight at 4°C with appropriate primary antibodies as follows: rabbit anti-mouse TLR4 antibody (Thermo Fisher, USA), rabbit anti-mouse myeloid differentiation factor 88 (MyD88) antibody (Santa Cruz, USA), rabbit anti-mouse TNFRassociated factor (TRAF6) antibody (Novus Biologicals, USA), rabbit anti-mouse NF-κB p65 antibody (GeneTex, USA), and rabbit anti-β-actin antibody (Proteintech, USA).

Techniques: Expressing, Binding Assay, Luciferase, Activity Assay, RNA Immunoprecipitation, Magnetic Beads, Control

Journal: PLoS Pathogens

Article Title: GPATCH3 negatively regulates RLR-mediated innate antiviral responses by disrupting the assembly of VISA signalosome

doi: 10.1371/journal.ppat.1006328

Figure Lengend Snippet: (A) 293T cells (2 x 10 6 ) were cotransfected with Flag-VISA (3 μg) and HA-tagged TBK1, TRAF6 or TRAF3 (3 μg), together with increasing amounts of GPATCH3 (0, 0.5 μg, 1.5μg). Coimmunoprecipitation and immunoblotting were performed with the indicated antibodies. (B) Wild-type or GPACH3-deficient cells (1.5 x 10 7 ) were left uninfected or infected with SeV for the indicated times. Coimmunoprecipitation and immunoblotting were performed with the indicated antibodies.

Article Snippet: HEK293T (Transformed Human Embryonic Kidney 293 cell line, ATCC) cells, A549 (Human Lung Adenocarcinoma cell line, ATCC) cells, PBMCs (Peripheral Blood Mononuclear Cells, Allcells), Human recombinant TNFα (R&D Systems), poly(I:C) (InvivoGen), Lipofectamine 2000 (Invitrogen), dual-specific luciferase assay kits (Promega), mouse monoclonal antibodies against Flag and β-actin (Sigma), HA (Origene), AIF, LMNB1 and TBK1 (Abcam), IRF-3 (Proteintech), VISA (Bethyl Laboratories), TRAF6 and GPATCH3 (Y-20) (Santa Cruz Biotechnology), phospho-IRF3 (Ser396), phospho-TBK1 (Ser172) and phospho-IKKα (Ser176)/β (Ser177) (Cell Signaling Technology) were purchased from the indicated companies.

Techniques: Western Blot, Infection

Journal: The Journal of Experimental Medicine

Article Title: miR-146a is a significant brake on autoimmunity, myeloproliferation, and cancer in mice

doi: 10.1084/jem.20101823

Figure Lengend Snippet: Exaggerated inflammatory response in miR-146a –null mice. (A and B) Serum levels of TNF (A) and IL-6 (B) in miR-146a −/− ( n = 5; KO) and WT ( n = 6) animals challenged intraperitoneally with sublethal dose of LPS (1 mg/kg). Peripheral blood was drawn at the times indicated in the graphs, and cytokine concentrations were assessed by ELISA. Results are shown as means; data are representative of two independent experiments. P-value calculations were performed using Student’s t test. (C) Kaplan-Meier survival curves of miR-146a −/− (KO) and WT mice ( n = 12 for each group) challenged with a lethal dose of LPS (35 mg/kg). Results are representative of two independent experiments. P-value calculation was performed using log-rank (Mantel-Cox) test. (D and E) Production of TNF (D) and IL-6 (E) by miR-146a −/− (KO) and WT BMDMs in response to 10 ng/ml LPS challenge. Cells were stimulated for the indicated time, and cytokine concentrations were assessed by ELISA. Data are shown as mean ± SD. Results are representative of three independent experiments. (F and G) Western blot analysis of TRAF6 and IRAK1 protein expression in WT and miR-146a −/− (KO) BMDMs (F) and CD19 + splenic B cells (G). Numbers under the blots denote relative expression normalized to β-actin expression for each sample. Two mice per genotype were analyzed. Results are representative of two independent experiments. (H and I) Quantification of Western blots in F and G. Relative TRAF6 and IRAK1 expression in WT and KO BMDMs (H) and CD19 + splenic B cells (I) is plotted. Data from two mice for each genotype were averaged and are expressed as a ratio of protein expression in miR-146a KO to WT cells. All experiments were carried in the B6/ miR-146a −/− strain of mice.

Article Snippet: Anti–mouse IRAK1 antibodies were obtained from Cell Signaling Technology, and anti-mouse TRAF6 were obtained from MBL International; anti–human IRAK1 and TRAF6 were purchased from Santa Cruz Biotechnology, Inc. Quantification of relative expression for each target protein was performed in Photoshop CS4 (Adobe), and β-actin levels were used for normalization.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

Journal: The Journal of Experimental Medicine

Article Title: miR-146a is a significant brake on autoimmunity, myeloproliferation, and cancer in mice

doi: 10.1084/jem.20101823

Figure Lengend Snippet: Ectopic expression of miR-146a in THP-1 cells results in attenuation of inflammatory response. (A and B) Production of TNF (A) and IL-6 (B) by THP-1 stable cell lines ectopically expressing either miR-146a (THP1/146) or scramble control sequence (THP1/SCR) in response to 1 µg/ml LPS stimulation. Cells were stimulated for the indicated time, and cytokines concentrations were assessed by ELISA. Results are shown as mean ± SD. Data are representative of two independent experiments. (C) Western blot analysis of TRAF6 and IRAK1 protein expression in THP1/146 and THP1/SCR clones stimulated or not with LPS, as indicated. Numbers under the blots denote relative expression normalized to β-actin expression for each sample. Data are representative of two independent experiments.

Article Snippet: Anti–mouse IRAK1 antibodies were obtained from Cell Signaling Technology, and anti-mouse TRAF6 were obtained from MBL International; anti–human IRAK1 and TRAF6 were purchased from Santa Cruz Biotechnology, Inc. Quantification of relative expression for each target protein was performed in Photoshop CS4 (Adobe), and β-actin levels were used for normalization.

Techniques: Expressing, Stable Transfection, Control, Sequencing, Enzyme-linked Immunosorbent Assay, Western Blot, Clone Assay

Journal: Cell reports

Article Title: A circRNA therapy based on Rnf103 to inhibit Vibrio anguillarum infection.

doi: 10.1016/j.celrep.2023.113314

Figure Lengend Snippet: Figure 1. Rnf103 inhibits innate immune response upon V anguillarum infection (A) Protein and mRNA levels of Rnf103 in MKCs after transfection with siRNA negative control (si-NC) or si-rnf103 (n = 3). (B) Bacterial plaque in MKCs after transfection with si-NC or si-rnf103 and with pcDNA3.1 or Rnf103 (n = 3; scale bars, 10 mm). (C) MKCs were transfected with NC or si-rnf103 and pcDNA3.1 or Rnf103 and infected with V. anguillarum (n = 3). (D) Luciferase activities were detected in epithelioma papulosum cyprini cells (EPCs) after cotransfection with Myd88, Tak1, P65, Nod1, and Traf6 expression plasmid, phRL-TK Renilla luciferase plasmid, luciferase reporters, and Rnf103 (n = 3). (E) Luciferase activities were detected in EPCs after cotransfection with phRL-TK Renilla luciferase plasmid, Traf6 luciferase reporters, and Rnf103 (n = 3). (F) Protein levels of Traf6 in MKCs after transfection with si-NC or si-rnf103 and with pcDNA3.1 or Rnf103 (n = 3). (G) qPCR assays were performed to determine the expression levels of the inflammatory factors in MKCs transfected with si-NC or si-rnf103 and pcDNA3.1 or Rnf103 after V. anguillarum infection (n = 3). Data are means ± SE. All data, including immunoblots, are representative of three or more independent experiments. *p < 0.05, **p < 0.01 by Student’s t test.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-M. miiuy Rnf103-177aa This paper N/A Rabbit anti-M. miiuy Rnf103 This paper N/A Rabbit anti-human Phospho-TAK1 Beyotime Cat# AF5863 Rabbit anti-human TRAF6 Boster Cat# BM4061 mouse anti-Flag Beyotime Cat# AF519 mouse anti-Myc Beyotime Cat# AF2864 mouse anti-HA Beyotime Cat# AF2858 mouse anti-Tubulin Beyotime Cat# AT819 HRP-conjugated anti-rabbit IgG Abbkine Cat# A25022 HRP-conjugated anti-mouse IgG Abbkine Cat# A25012 Bacterial and virus strains Vibrio anguillarum This paper N/A Chemicals, peptides, and recombinant proteins DAPI Beyotime Cat# C1002 CHX MedChemExpress Cat# HY-12320 Carfilzomib Beyotime Cat# SF4157 Bortzeomib Beyotime Cat# SC0263 RNase R Beyotime Cat# R7092S T4 Polynucleotide Kinase Beyotime Cat# D7096 T4 RNA Ligase 2 Beyotime Cat# R0635S T7 RNA Polymerase Beyotime Cat# D7069 Critical commercial assays Lipofectamine RNAiMAX Invitrogen Cat# 13778150 Lipofectamine 3000 Invitrogen Cat# L3000015 FastQuant RT Kit Tiangen Cat# KR106-03 Magna RIP RNA-Binding Protein Immunoprecipitation Kit Millipore Cat# 17-700 BCA Protein Assay kit Beyotime Cat# P0012S CellTiter-Glo Luminescent Cell Viability assays Kit Promega Cat# G7570 Mut Express II Fast Mutagenesis Kit V2 Vazyme Cat# C214 Endotoxin-Free Plasmid DNA Miniprep Kit Tiangen Cat# DP118 SYBR Premix Ex TaqTM Takara Cat# DRR041S Deposited data RNA sequencing data This paper GenBank accession number: PRJNA691457 and PRJNA846999 Experimental models: Cell lines EPC ATCC Cat# CRL-2872 MKC This paper N/A Experimental models: Organisms M. miiuy ( 50 g, six-weeks-old, male or female) This paper N/A Danio rerio (four-mouths-old, male or female) This paper N/A Oligonucleotides Primers are listed in Table S1 This paper N/A (Continued on next page) 14 Cell Reports 42, 113314, November 28, 2023

Techniques: Infection, Transfection, Negative Control, Luciferase, Cotransfection, Expressing, Plasmid Preparation, Western Blot

Journal: Cell reports

Article Title: A circRNA therapy based on Rnf103 to inhibit Vibrio anguillarum infection.

doi: 10.1016/j.celrep.2023.113314

Figure Lengend Snippet: Figure 2. Rnf103 promotes the proteasome degradation of Traf6 (A) MKCs were transfected with FLAG-Rnf103 plasmids or si-rnf103 and treated with 10 mM cyclohexide (CHX) after V. anguillarum infection (n = 3). (B) MKCs were transfected with FLAG-Rnf103 and treated with DMSO or 10 mM MG132 or carfilzomib or bortzeomib after V. anguillarum infection (n = 3). (C) FLAG-Rnf103 was transfected into MKCs (n = 3; scale bars, 10 mm). (D) Immunoprecipitation and immunoblot analysis of FLAG-Rnf103 in MKCs (n = 3). (E) Immunoprecipitation of FLAG-Rnf103, with Traf6 and Traf6 mutant in MKCs. (F) Coimmunoprecipitation analysis of Traf6 ubiquitination in MKC cotransfected with FLAG-Rnf103 plasmid and HA-ubiquitin-WT, HA-ubiquitin-K11, HA- ubiquitin-K27, HA-ubiquitin-K48, or HA-ubiquitin-K63 plasmids (n = 3). Data are means ± SE. All data, including immunoblots, are representative of three or more independent experiments.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-M. miiuy Rnf103-177aa This paper N/A Rabbit anti-M. miiuy Rnf103 This paper N/A Rabbit anti-human Phospho-TAK1 Beyotime Cat# AF5863 Rabbit anti-human TRAF6 Boster Cat# BM4061 mouse anti-Flag Beyotime Cat# AF519 mouse anti-Myc Beyotime Cat# AF2864 mouse anti-HA Beyotime Cat# AF2858 mouse anti-Tubulin Beyotime Cat# AT819 HRP-conjugated anti-rabbit IgG Abbkine Cat# A25022 HRP-conjugated anti-mouse IgG Abbkine Cat# A25012 Bacterial and virus strains Vibrio anguillarum This paper N/A Chemicals, peptides, and recombinant proteins DAPI Beyotime Cat# C1002 CHX MedChemExpress Cat# HY-12320 Carfilzomib Beyotime Cat# SF4157 Bortzeomib Beyotime Cat# SC0263 RNase R Beyotime Cat# R7092S T4 Polynucleotide Kinase Beyotime Cat# D7096 T4 RNA Ligase 2 Beyotime Cat# R0635S T7 RNA Polymerase Beyotime Cat# D7069 Critical commercial assays Lipofectamine RNAiMAX Invitrogen Cat# 13778150 Lipofectamine 3000 Invitrogen Cat# L3000015 FastQuant RT Kit Tiangen Cat# KR106-03 Magna RIP RNA-Binding Protein Immunoprecipitation Kit Millipore Cat# 17-700 BCA Protein Assay kit Beyotime Cat# P0012S CellTiter-Glo Luminescent Cell Viability assays Kit Promega Cat# G7570 Mut Express II Fast Mutagenesis Kit V2 Vazyme Cat# C214 Endotoxin-Free Plasmid DNA Miniprep Kit Tiangen Cat# DP118 SYBR Premix Ex TaqTM Takara Cat# DRR041S Deposited data RNA sequencing data This paper GenBank accession number: PRJNA691457 and PRJNA846999 Experimental models: Cell lines EPC ATCC Cat# CRL-2872 MKC This paper N/A Experimental models: Organisms M. miiuy ( 50 g, six-weeks-old, male or female) This paper N/A Danio rerio (four-mouths-old, male or female) This paper N/A Oligonucleotides Primers are listed in Table S1 This paper N/A (Continued on next page) 14 Cell Reports 42, 113314, November 28, 2023

Techniques: Transfection, Infection, Immunoprecipitation, Western Blot, Mutagenesis, Ubiquitin Proteomics, Plasmid Preparation

Journal: Cell reports

Article Title: A circRNA therapy based on Rnf103 to inhibit Vibrio anguillarum infection.

doi: 10.1016/j.celrep.2023.113314

Figure Lengend Snippet: Figure 4. Rnf103-177aa inhibits V. anguillarum infection by competing with Rnf103 to bind Traf6 (A) The schematic diagram of siRNAs. (B) qPCR analysis of circRnf103 and Rnf103 mRNA in MKCs treated with siRNAs and circRnf103 and circRnf103-ATG-mut (n = 3). (C) Bacterial plaque in MKCs after transfection with si-NC or si-circRnf103 and circ-NC or circRnf103 (n = 3; scale bars, 10 mm). (D) MKCs were transfected with si-NC, si-circRnf103, circ-NC, circRnf103, circRnf103-ATG-mut, or circRnf103-IRES-mut and then infected with V. anguillarum (n = 3). (E) Luciferase activities were detected in EPC after cotransfection with Myd88, Tak1, p65, Nod1, and Traf6 expression plasmid, phRL-TK Renilla luciferase plasmid, luciferase reporters, circRnf103, or circRnf103-ATG-mut (n = 3). (F) Luciferase activities were detected in EPC after cotransfection with phRL-TK Renilla luciferase plasmid, Traf6 luciferase reporters, circRnf103, or circRnf103- ATG-mut (n = 3). (G) Protein levels of Traf6 in MKCs after transfection with si-NC, si-circRnf103, circ-NC, circRnf103, or circRnf103-ATG-mut (n = 3). (H) Immunoprecipitation and immunoblot analysis of FLAG-circRnf103 in MKCs (n = 3). (I) FLAG-circRnf103 was transfected into MKCs (n = 3; scale bars, 10 mm). (J) Immunoprecipitation of FLAG-circRnf103, with Traf6 and Traf6 mutant in MKCs (n = 3). (K) Protein levels of Traf6 in MKCs after cotransfection with Rnf103 and circRnf103 (n = 3). (L) Coimmunoprecipitation analysis of Traf6 ubiquitination in MKCs cotransfected with FLAG-Rnf103 expression plasmid and FLAG-circRnf103-P plasmid and HA-ubiquitin-WT, HA-ubiquitin-K48 plasmids (n = 3). (M) Upper: schematic diagram of Rnf103-D(209-349aa) plasmid construction. Lower: immunoprecipitation of FLAG-Rnf103, FLAG-Rnf103-D(209-349aa), in MKCs (n = 3). Data are means ± SE. All data, including immunoblots, are representative of three or more independent experiments. *p < 0.05, **p < 0.01 by Student’s t test.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-M. miiuy Rnf103-177aa This paper N/A Rabbit anti-M. miiuy Rnf103 This paper N/A Rabbit anti-human Phospho-TAK1 Beyotime Cat# AF5863 Rabbit anti-human TRAF6 Boster Cat# BM4061 mouse anti-Flag Beyotime Cat# AF519 mouse anti-Myc Beyotime Cat# AF2864 mouse anti-HA Beyotime Cat# AF2858 mouse anti-Tubulin Beyotime Cat# AT819 HRP-conjugated anti-rabbit IgG Abbkine Cat# A25022 HRP-conjugated anti-mouse IgG Abbkine Cat# A25012 Bacterial and virus strains Vibrio anguillarum This paper N/A Chemicals, peptides, and recombinant proteins DAPI Beyotime Cat# C1002 CHX MedChemExpress Cat# HY-12320 Carfilzomib Beyotime Cat# SF4157 Bortzeomib Beyotime Cat# SC0263 RNase R Beyotime Cat# R7092S T4 Polynucleotide Kinase Beyotime Cat# D7096 T4 RNA Ligase 2 Beyotime Cat# R0635S T7 RNA Polymerase Beyotime Cat# D7069 Critical commercial assays Lipofectamine RNAiMAX Invitrogen Cat# 13778150 Lipofectamine 3000 Invitrogen Cat# L3000015 FastQuant RT Kit Tiangen Cat# KR106-03 Magna RIP RNA-Binding Protein Immunoprecipitation Kit Millipore Cat# 17-700 BCA Protein Assay kit Beyotime Cat# P0012S CellTiter-Glo Luminescent Cell Viability assays Kit Promega Cat# G7570 Mut Express II Fast Mutagenesis Kit V2 Vazyme Cat# C214 Endotoxin-Free Plasmid DNA Miniprep Kit Tiangen Cat# DP118 SYBR Premix Ex TaqTM Takara Cat# DRR041S Deposited data RNA sequencing data This paper GenBank accession number: PRJNA691457 and PRJNA846999 Experimental models: Cell lines EPC ATCC Cat# CRL-2872 MKC This paper N/A Experimental models: Organisms M. miiuy ( 50 g, six-weeks-old, male or female) This paper N/A Danio rerio (four-mouths-old, male or female) This paper N/A Oligonucleotides Primers are listed in Table S1 This paper N/A (Continued on next page) 14 Cell Reports 42, 113314, November 28, 2023

Techniques: Infection, Transfection, Luciferase, Cotransfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Mutagenesis, Ubiquitin Proteomics