pduo plasmids  (InvivoGen)

Journal: Frontiers in Immunology

Article Title: Cell membrane-bound toll-like receptor-1/2/4/6 monomers and -2 heterodimer inhibit enterovirus 71 replication by activating the antiviral innate response

doi: 10.3389/fimmu.2023.1187035

Figure Lengend Snippet: EV71 infection activates the antiviral innate immunity via toll-like receptor (TLR) monomers and TLR2 heterodimers to limit its replication. (A) Human-derived TLR2 and TLR2 heterodimer (TLR2/TLR1 and TLR2/TLR6) or (B) mouse-derived TLR2 heterodimer (mTLR2/mTLR1 and mTLR2/mTLR6) plasmids were transfected into HEK293 cells at a dose of 1 or 2 μg for 24 h, followed by infection with EV71 at a multiplicity of infection (MOI) of 1 for 24 (h) n = 3. (C) Human-derived TLR1, TLR6, and TLR2/CD14 or (D) mouse-derived mTLR2, mTLR1, and mTLR6 plasmids were transfected into HEK293 cells at a dose of 1 μg for 24 h, followed by infection with EV71 at an MOI of 1 for 24 (h) (E) Human-derived TLR2 and TLR2 heterodimer (TLR2/TLR1 and TLR2/TLR6) plasmids were transfected into rhabdomyosarcoma (RD) cells for 24 h and infected with EV71 at an MOI of 0.5 or 1 for 24 (h) Genome copies of EV71 were determined using quantitative polymerase chain reaction (qPCR), and relative fold differences compared with HEK293 cells infected with EV71 for 24 h were calculated. n = 3. (F) Human-derived TLR2 heterodimer (TLR2/TLR1, TLR2/TLR6, and TLR2/CD14) or (G) mouse-derived TLR2 heterodimer (mTLR2/mTLR1 and mTLR2/mTLR6) plasmids were transfected into HEK293 cells at a dose of 1 or 2 μg for 24 h, followed by infection with EV71 at an MOI of 1 for 24 (h) Supernatants were collected and interleukin (IL)-8 concentrations were determined using an enzyme-linked immunosorbent assay (ELISA) kit. n = 3. (H) Human-derived TLR monomer (TLR2, TLR1, and TLR6) and TLR2 heterodimer (TLR2/TLR1, TLR2/TLR6, and TLR2/CD14) plasmids or (I) mouse-derived TLR monomer (mTLR2, mTLR1, and mTLR6) and TLR2 heterodimer (mTLR2/mTLR1 and mTLR2/mTLR6) plasmids were transfected into HEK293 cells at a dose of 1 μg for 24 h, followed by infection with EV71 at an MOI of 1 for 24 (h) Total proteins were collected, and activation of the phosphoinositide 3-kinase/protein kinase B ( PI3K/AKT ) and mitogen-activated protein kinase ( MAPK ) pathways was assessed via western blotting. n = 3. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: All these plasmids were purchased from Addgene ( https://www.addgene.org ). mTLR6 plasmid pUNO1-mTLR06-HA3x (puno1ha-mtlr6), TLR2 heterodimer plasmids, including pDUO-hTLR6/TLR2 (pduo-htlr6tlr2), pDUO-hTLR1/TLR2 (pduo-htlr1tlr2), and pDUO-hCD14/TLR2 (pduo-hcd14tlr2) plasmids, TLR1/2/4/6 Dominant-negative TIR-less (DN [ΔTIR]) plasmids, pUNO1-hTLR1-DN-HA (puno1ha-htlr1-dn), pUNO1-hTLR2-DN-HA (puno1ha-htlr2-dn), pUNO1-hTLR4-DN-HA (puno1ha-htlr4a-dn), and pUNO1-hTLR6-DN-HA (puno1ha-htlr6-dn), were purchased from InvivoGen (San Diego, CA, USA).

Techniques: Infection, Derivative Assay, Transfection, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Activation Assay, Western Blot

Journal: Frontiers in Immunology

Article Title: Cell membrane-bound toll-like receptor-1/2/4/6 monomers and -2 heterodimer inhibit enterovirus 71 replication by activating the antiviral innate response

doi: 10.3389/fimmu.2023.1187035

Figure Lengend Snippet: EV71 replication is inhibited by conditional changes in TLR2 heterodimers. (A) Human–mouse chimeric TLR2 heterodimer (mTLR2/TLR1, TLR2/mTLR1, mTLR2/TLR6, and TLR2/mTLR6) plasmids and (B) single dominant-negative TIR-less (DN) TLR2 heterodimer (DN-TLR2/TLR1, TLR2/DN-TLR1, DN-TLR2/TLR6, and TLR2/DN-TLR6) plasmids were transfected into HEK293 cells at a dose of 1 μg for 24 h, followed by infection with EV71 at an MOI of 1 for 24 (h) DN-TLR (DN-TLR1, DN-TLR2, and DN-TLR6) plasmids were transfected into (C) HEK293 or (D) UT-SCC-60B cells at a dose of 1 μg for 24 h, followed by infection with EV71 at an MOI of 1 for 24 (h) n = 3. Genome copies of EV71 were determined and relative fold differences were calculated. (E) Human–mouse chimeric TLR2 heterodimer (mTLR2/TLR1, TLR2/mTLR1, mTLR2/TLR6, and TLR2/mTLR6) plasmids were transfected into HEK293 cells at a dose of 1 or 2 μg for 24 h, followed by infection with EV71 at an MOI of 1 for 24 (h) n = 3. (F) Concentrations of IL-8 in the supernatant of group (B) . n = 3. (G) Human–mouse chimeric TLR2 heterodimer (mTLR2/TLR1, TLR2/mTLR1, mTLR2/TLR6, and TLR2/mTLR6) plasmids or (H) single dominant-negative TIR-less TLR2 heterodimer (DN-TLR2/TLR1 and TLR2/DN-TLR1) plasmids were transfected into HEK293 cells at a dose of 1 μg for 24 h, followed by infection with EV71 at an MOI of 1 for 24 (h) Total proteins were collected, and activation of the PI3K/AKT and MAPK pathways was assessed via western blotting. n = 3. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: All these plasmids were purchased from Addgene ( https://www.addgene.org ). mTLR6 plasmid pUNO1-mTLR06-HA3x (puno1ha-mtlr6), TLR2 heterodimer plasmids, including pDUO-hTLR6/TLR2 (pduo-htlr6tlr2), pDUO-hTLR1/TLR2 (pduo-htlr1tlr2), and pDUO-hCD14/TLR2 (pduo-hcd14tlr2) plasmids, TLR1/2/4/6 Dominant-negative TIR-less (DN [ΔTIR]) plasmids, pUNO1-hTLR1-DN-HA (puno1ha-htlr1-dn), pUNO1-hTLR2-DN-HA (puno1ha-htlr2-dn), pUNO1-hTLR4-DN-HA (puno1ha-htlr4a-dn), and pUNO1-hTLR6-DN-HA (puno1ha-htlr6-dn), were purchased from InvivoGen (San Diego, CA, USA).

Techniques: Dominant Negative Mutation, Transfection, Infection, Activation Assay, Western Blot

Journal: Frontiers in Immunology

Article Title: Cell membrane-bound toll-like receptor-1/2/4/6 monomers and -2 heterodimer inhibit enterovirus 71 replication by activating the antiviral innate response

doi: 10.3389/fimmu.2023.1187035

Figure Lengend Snippet: EV71 infection activates antiviral innate immunity via TLR4 and TLR2/TLR4 to limit its replication. (A) Human-derived TLR4 and TLR2/TLR4 plasmids or (B) mouse-derived mTLR4 and mTLR2/mTLR4 plasmids were transfected into HEK293 cells at a dose of 1 μg for 24 h, followed by infection with EV71 at an MOI of 1 for 24 (h) n = 3. (C) Human-derived TLR4 and TLR2/TLR4 plasmids were transfected into RD cells at a dose of 1 μg for 24 h, followed by infection with EV71 at an MOI of 0.5 or 1 for 24 (h) n = 3. Genome copies of EV71 were determined and relative fold differences were calculated. (D) Human-derived TLR4 and TLR2/TLR4 plasmids were transfected into HEK293 cells at a dose of 1 or 2 μg for 24 h, followed by infection with EV71 at an MOI of 1 for 24 (h) n = 3. (E) Concentrations of IL-8 in the supernatant of group (B) were determined. (F) Human-derived TLR2, TLR4, and TLR2/TLR4 plasmids or (G) mouse-derived mTLR2, mTLR4, and mTLR2/mTLR4 plasmids were transfected into HEK293 cells at a dose of 1 μg for 24 h, followed by infection with EV71 at an MOI of 1 for 24 (h) n = 3. Total proteins were collected, and activation of the PI3K/AKT and MAPK pathways was assessed via western blotting. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: All these plasmids were purchased from Addgene ( https://www.addgene.org ). mTLR6 plasmid pUNO1-mTLR06-HA3x (puno1ha-mtlr6), TLR2 heterodimer plasmids, including pDUO-hTLR6/TLR2 (pduo-htlr6tlr2), pDUO-hTLR1/TLR2 (pduo-htlr1tlr2), and pDUO-hCD14/TLR2 (pduo-hcd14tlr2) plasmids, TLR1/2/4/6 Dominant-negative TIR-less (DN [ΔTIR]) plasmids, pUNO1-hTLR1-DN-HA (puno1ha-htlr1-dn), pUNO1-hTLR2-DN-HA (puno1ha-htlr2-dn), pUNO1-hTLR4-DN-HA (puno1ha-htlr4a-dn), and pUNO1-hTLR6-DN-HA (puno1ha-htlr6-dn), were purchased from InvivoGen (San Diego, CA, USA).

Techniques: Infection, Derivative Assay, Transfection, Activation Assay, Western Blot

Journal: Frontiers in Immunology

Article Title: Cell membrane-bound toll-like receptor-1/2/4/6 monomers and -2 heterodimer inhibit enterovirus 71 replication by activating the antiviral innate response

doi: 10.3389/fimmu.2023.1187035

Figure Lengend Snippet: Conditional changes in TLR4 and TLR2/TLR4 heterodimers inhibit EV71 replication by activating innate immunity. (A) Human–mouse chimeric TLR2 heterodimer (mTLR2/TLR4 and TLR2/mTLR4) plasmids were transfected into HEK293 cells at a dose of 1 or 2 μg for 24 h, followed by infection with EV71 at an MOI of 1 for 24 (h) n = 3. (B) Single dominant-negative TIR-less TLR2/TLR4 heterodimer (DN-TLR2/TLR4 and TLR2/DN-TLR4) plasmids were transfected into HEK293 cells at a dose of 1 μg for 24 h, followed by infection with EV71 at an MOI of 1 for 24 (h) Single dominant-negative TIR-less TLR4 plasmid was transfected into (C) HEK293 or (D) UT-SCC-60B cells at a dose of 1 μg for 24 h, followed by infection with EV71 at an MOI of 1 for 24 (h) n = 3. (E) TLR4 single nucleotide mutation plasmids (A896G, C1196T, and C2141A) were transfected into HEK293 cells at a dose of 1 μg for 24 h, followed by infection with EV71 at an MOI of 1 for 24 (h) n = 3. Genome copies of EV71 were determined and relative fold differences were calculated. Concentrations of IL-8 in the supernatants of (F) human–mouse chimeric TLR2 heterodimer and (G) single dominant-negative TIR-less TLR2 heterodimer groups were determined. n = 3. Activation of the PI3K/AKT and MAPK pathways in (H) human–mouse chimeric TLR2 heterodimers and (I) DN-TLR2/TLR4 groups was assessed via western blotting. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: All these plasmids were purchased from Addgene ( https://www.addgene.org ). mTLR6 plasmid pUNO1-mTLR06-HA3x (puno1ha-mtlr6), TLR2 heterodimer plasmids, including pDUO-hTLR6/TLR2 (pduo-htlr6tlr2), pDUO-hTLR1/TLR2 (pduo-htlr1tlr2), and pDUO-hCD14/TLR2 (pduo-hcd14tlr2) plasmids, TLR1/2/4/6 Dominant-negative TIR-less (DN [ΔTIR]) plasmids, pUNO1-hTLR1-DN-HA (puno1ha-htlr1-dn), pUNO1-hTLR2-DN-HA (puno1ha-htlr2-dn), pUNO1-hTLR4-DN-HA (puno1ha-htlr4a-dn), and pUNO1-hTLR6-DN-HA (puno1ha-htlr6-dn), were purchased from InvivoGen (San Diego, CA, USA).

Techniques: Transfection, Infection, Dominant Negative Mutation, Plasmid Preparation, Mutagenesis, Activation Assay, Western Blot

Journal: Frontiers in Immunology

Article Title: Cell membrane-bound toll-like receptor-1/2/4/6 monomers and -2 heterodimer inhibit enterovirus 71 replication by activating the antiviral innate response

doi: 10.3389/fimmu.2023.1187035

Figure Lengend Snippet: EV71 capsid proteins induce cytokine responses via TLR2 and TLR2 heterodimers. UT-SCC-60B cells were stimulated with purified recombinant prokaryotic-expressed EV71 capsid proteins at a final concentration of 80 μg/mL, or EV71 capsid recombinant eukaryotic plasmids were transfected into UT-SCC-60B cells at a dose of 1 μg for 24 (h) n = 3. Concentrations of IL-6 and IL-8 in (A) recombinant EV71 capsid protein stimulated and (B) EV71 capsid plasmid transfected groups were determined. Activation of the PI3K/AKT and MAPK pathways in (C) recombinant EV71 capsid protein stimulated and (D) EV71 capsid plasmid transfected groups were assessed via western blotting. Human-derived TLR2 and TLR2 heterodimer (TLR2/TLR1 and TLR2/TLR6) plasmids were transfected into HEK293 cells at a dose of 1 μg for 24 h and stimulated with recombinant EV71 capsid proteins at a final concentration of 80 μg/mL for 24 (h) n = 3. (E) Concentrations of IL-8 and (G) activation of the PI3K/AKT and MAPK pathways were determined. Human-derived TLR2 and TLR2 heterodimer (TLR2/TLR1 and TLR2/TLR6) and EV71 capsid plasmids were co-transfected into UT-SCC-60B cells at a dose of 1 μg for 24 (h) n = 3. (F) Concentrations of IL-8 and (H) activation of the PI3K/AKT and MAPK pathways were determined. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: All these plasmids were purchased from Addgene ( https://www.addgene.org ). mTLR6 plasmid pUNO1-mTLR06-HA3x (puno1ha-mtlr6), TLR2 heterodimer plasmids, including pDUO-hTLR6/TLR2 (pduo-htlr6tlr2), pDUO-hTLR1/TLR2 (pduo-htlr1tlr2), and pDUO-hCD14/TLR2 (pduo-hcd14tlr2) plasmids, TLR1/2/4/6 Dominant-negative TIR-less (DN [ΔTIR]) plasmids, pUNO1-hTLR1-DN-HA (puno1ha-htlr1-dn), pUNO1-hTLR2-DN-HA (puno1ha-htlr2-dn), pUNO1-hTLR4-DN-HA (puno1ha-htlr4a-dn), and pUNO1-hTLR6-DN-HA (puno1ha-htlr6-dn), were purchased from InvivoGen (San Diego, CA, USA).

Techniques: Purification, Recombinant, Concentration Assay, Transfection, Plasmid Preparation, Activation Assay, Western Blot, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Cell membrane-bound toll-like receptor-1/2/4/6 monomers and -2 heterodimer inhibit enterovirus 71 replication by activating the antiviral innate response

doi: 10.3389/fimmu.2023.1187035

Figure Lengend Snippet: EV71 capsid proteins induce cytokine responses via TLR4 and TLR2/TLR4 heterodimer. Human-derived TLR4 and TLR2/TLR4 plasmids were transfected into HEK293 cells at a dose of 1 μg for 24 h, followed by stimulation with recombinant EV71 capsid proteins at a final concentration of 80 μg/mL for 24 (h) n = 3. (A) Concentrations of IL-8 and (C) activation of the PI3K/AKT and MAPK pathways were determined. Human-derived TLR4 and TLR2/TLR4 and EV71 capsid plasmids were co-transfected into UT-SCC-60B cells at a dose of 1 μg for 24 (h) n = 3. (B) Concentrations of IL-8 and (D) activation of the PI3K/AKT and MAPK pathways were determined. (E) Schematic model for the activation of innate immunity by EV71 and capsid proteins via cell membrane-bound TLR1/2/4/6 monomers and TLR2 heterodimers. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: All these plasmids were purchased from Addgene ( https://www.addgene.org ). mTLR6 plasmid pUNO1-mTLR06-HA3x (puno1ha-mtlr6), TLR2 heterodimer plasmids, including pDUO-hTLR6/TLR2 (pduo-htlr6tlr2), pDUO-hTLR1/TLR2 (pduo-htlr1tlr2), and pDUO-hCD14/TLR2 (pduo-hcd14tlr2) plasmids, TLR1/2/4/6 Dominant-negative TIR-less (DN [ΔTIR]) plasmids, pUNO1-hTLR1-DN-HA (puno1ha-htlr1-dn), pUNO1-hTLR2-DN-HA (puno1ha-htlr2-dn), pUNO1-hTLR4-DN-HA (puno1ha-htlr4a-dn), and pUNO1-hTLR6-DN-HA (puno1ha-htlr6-dn), were purchased from InvivoGen (San Diego, CA, USA).

Techniques: Derivative Assay, Transfection, Recombinant, Concentration Assay, Activation Assay, Membrane

Journal: Vaccines

Article Title: Type IIb Heat Labile Enterotoxin B Subunit as a Mucosal Adjuvant to Enhance Protective Immunity against H5N1 Avian Influenza Viruses

doi: 10.3390/vaccines8040710

Figure Lengend Snippet: Recombinant LTIIb-B5 protein expression, purification, and characterization ( A ) Left panel, purified LTIIb-B5 protein with Coomassie blue staining; right panel, Western blot. Pentameric LTIIb-B5 is located at ~50 kDa, monomeric LTIIb-B5 at ~12 kDa (denatured). ( B ) Pentameric and monomeric LTIIb-B5 formation under different biochemical conditions. Boiled proteins were heated to 100 °C for 5 min. ( C ) FPLC size-exclusion elution profile for LTIIb-B5 (eluted at 55 mL, corresponding to ~70 kDa). Four marker proteins are Ribonuclease (13.7k), Chymotrypsinogen (25k), Ovalbumin (43k) and Albumin (66.5k). Results indicate the presence of LTIIb-B5 pentamers in solution. ( D ) 1 H- 15 N HSQC spectrum of LTIIb-B5. Peak dispersion indicates dynamic folded structure. ( E ) TLR2/1 signal functional assay results for LTIIb-B5. HEK 293A cells contained over-expressed hTLR1/hTLR2 receptor and NF-kB-driven luciferase due to transient transfection. Transfected cells were treated with diluted LTIIb-B5 and Pam3CSK4 (1 pg/mL to 10 μg/mL) and held for 5 h at 37 °C. TLR2/1 activity was detected concurrently with luciferase activity.

Article Snippet: HEK 293A cells were seeded and held overnight in culture dishes (6 × 10 6 cells/dish) prior to co-transfection with pDUO-hTLR1/hTLR2 plasmids (InvivoGen, San Diego, CA, USA) (7.5 μg/dish) and pGL4.32 (luc2p/NF-κB-RE/Hygro) vectors (Promega) (3 μg/dish) using Turbofect (Thermo Fisher Scientific, Waltham, IL, USA) transfection reagent.

Techniques: Recombinant, Expressing, Purification, Staining, Western Blot, Marker, Dispersion, Functional Assay, Luciferase, Transfection, Activity Assay