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: 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

Journal: Neural Regeneration Research

Article Title: An enriched environment reduces hippocampal inflammatory response and improves cognitive function in a mouse model of stroke

doi: 10.4103/1673-5374.338999

Figure Lengend Snippet: Flow chart of this study . SE group: Stroke + standard environment group; EE group: enriched environment group; IE group: stroke + isolated environment group; HE: hematoxylin-eosin; TRAF6: factor receptor-associated factor 6; TNF-α: tumor necrosis factor-α; IL-6: interleukin 6; NF-κBp65: nuclear factor kappa-Bp65; TTC: triphenyl tetrazolium chloride; RT-qPCR: real time quantitative polymerase chain reaction.

Article Snippet: The primary antibodies used were anti-NF-κBp65 (1:1000, rabbit, Abcam, Cat# ab76302, RRID: AB_1524028), anti-TRAF6 (1:2000, rabbit, Abcam, Cat# ab33915, RRID: AB_778572), anti-TNF-α (1:1000, rabbit, Abcam, Cat# ab183218, RRID: AB_2889388), and anti-IL-6 (1:1000, rabbit, Abcam, Cat# ab229381, RRID: AB_2861234).

Techniques: Isolation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: Neural Regeneration Research

Article Title: An enriched environment reduces hippocampal inflammatory response and improves cognitive function in a mouse model of stroke

doi: 10.4103/1673-5374.338999

Figure Lengend Snippet: Effects of the enriched environment on the protein expression levels of TRAF6, NF-κBp65 (nucleus), TNF-α, and IL-6 in the hippocampus of a mouse model of stroke using Western blot assay . (A) Bands of TRAF6, TNF-α, and IL-6 protein. (B) Bands of NF-κBp65 (nucleus) protein. (C–F) Quantitative results of TRAF6, TNF-α, IL-6, and NF-κBp65 (nucleus) protein expression. The expression of TRAF6, TNF-α, and IL-6 protein was normalized against GAPDH, and the expression of NF-κBp65 (nucleus) protein was normalized against Histone H3. Data are expressed as the mean ± SD ( n = 6). * P < 0.05, vs . sham group; # P < 0.05, vs . SE group (one-way analysis of variance followed by least significant difference test). EE group: stroke + enriched environment group; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IE group: stroke + isolated environment group; IL-6: interleukin 6; NF-κBp65: nuclear factor kappa-Bp65; SE group: stroke + standard environment group; TNF-α: tumor necrosis factor-α; TRAF6: factor receptor-associated factor 6.

Article Snippet: The primary antibodies used were anti-NF-κBp65 (1:1000, rabbit, Abcam, Cat# ab76302, RRID: AB_1524028), anti-TRAF6 (1:2000, rabbit, Abcam, Cat# ab33915, RRID: AB_778572), anti-TNF-α (1:1000, rabbit, Abcam, Cat# ab183218, RRID: AB_2889388), and anti-IL-6 (1:1000, rabbit, Abcam, Cat# ab229381, RRID: AB_2861234).

Techniques: Expressing, Western Blot, Isolation

Journal: Neural Regeneration Research

Article Title: An enriched environment reduces hippocampal inflammatory response and improves cognitive function in a mouse model of stroke

doi: 10.4103/1673-5374.338999

Figure Lengend Snippet: Effects of the enriched environment on the expression levels of miR-146a-5p (A) and TRAF6 mRNA (B) in the hippocampus of a mouse model of stroke using quantitative reverse transcription-polymerase chain reaction . Data are expressed as the mean ± SD ( n = 6), and were analyzed using nonparametric tests. * P < 0.05, vs . sham group; # P < 0.05, vs . SE group (Kruskal-Wallis test followed by Mann-Whitney U test). EE group: stroke + enriched environment group; IE group: stroke + isolated environment group; SE group: stroke + standard environment group; TRAF6: tumor necrosis factor receptor-associated factor 6.

Article Snippet: The primary antibodies used were anti-NF-κBp65 (1:1000, rabbit, Abcam, Cat# ab76302, RRID: AB_1524028), anti-TRAF6 (1:2000, rabbit, Abcam, Cat# ab33915, RRID: AB_778572), anti-TNF-α (1:1000, rabbit, Abcam, Cat# ab183218, RRID: AB_2889388), and anti-IL-6 (1:1000, rabbit, Abcam, Cat# ab229381, RRID: AB_2861234).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY, Isolation

Journal: Neural Regeneration Research

Article Title: An enriched environment reduces hippocampal inflammatory response and improves cognitive function in a mouse model of stroke

doi: 10.4103/1673-5374.338999

Figure Lengend Snippet: TRAF6 as a target gene of miR-146a-5p according to the database prediction comparison analysis . There were three potential binding sites of the TRAF6 mRNA 3′ untranslated region to miR-146a-5p according to the principle of base complementary pairing (A=T, C=G). TRAF6: Tumor necrosis factor receptor-associated factor 6.

Article Snippet: The primary antibodies used were anti-NF-κBp65 (1:1000, rabbit, Abcam, Cat# ab76302, RRID: AB_1524028), anti-TRAF6 (1:2000, rabbit, Abcam, Cat# ab33915, RRID: AB_778572), anti-TNF-α (1:1000, rabbit, Abcam, Cat# ab183218, RRID: AB_2889388), and anti-IL-6 (1:1000, rabbit, Abcam, Cat# ab229381, RRID: AB_2861234).

Techniques: Binding Assay