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recombinant mbp  (New England Biolabs)


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    Structured Review

    New England Biolabs recombinant mbp
    Recombinant Mbp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 7578 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/recombinant+mbp/Amylose+Resin/pmc12679992-303-14-22
    Average 99 stars, based on 7578 article reviews
    recombinant mbp - by Bioz Stars, 2026-10
    99/100 stars

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    Recombinant:

    Article Title: SNHG15-Mediated Localization of Nucleolin at the Cell Protrusions Regulates CDH2 mRNA Expression and Cell Invasion.
    Article Snippet: .. Briefly, recombinant MBP–MCP-conjugated amylose resin (NEB, USA) was incubated with cell lysates prepared from cells expressing MS2-tagged full-length or truncated SNHG15 at 4 ◦C for 4 h in the presence of RNase and protease inhibitors. ..

    Article Title: XerC-mediated DNA inversion at the inverted repeats of the UU172-phase-variable element of Ureaplasma parvum serovar 3.
    Article Snippet: Zimmerman et al. / Microb vent was monitored by restriction analysis of the plasmids conaining the synthetic phase-variable element. aterials and methods ultivation and transformation of microorganisms E. coli Top10 F′ (F ́{lacIq Tn10 (TetR)} mcrA (mrr-hsdRMScrBC) 80lacZ M15 lacX74 recA1 araD139 (ara-leu)7697 alU galK rpsL endA1 nupG) (LifeTechnologies) was grown in broth r on agar containing 10 g L−1 trypton, 10 g L−1 yeast extract and g L−1 NaCl. .. Competent cells were transformed by heat shock at 3 ◦C for 2 min. urification of recombinant proteins The recombinant MBP (maltose binding protein) fusion proein MBP::XerC-6×His was purified as described (Zimmerman t al. 2013) by affinity chromatography over amylose resin accordng to the manufacturer’s protocol (NEB; http://www.neb.com/ ebecomm/manualfiles/manuale8000.pdf, Affinity Chromatograhy, Method I). .. The elution was then further purified over i2+-charged ProBondTM Resin (LifeTechnologies) and eluted with uffer (50 mM Tris, 300 mM NaCl, pH 7.5). lectrophoretic mobility shift assay (EMSA) EMSA analysis was carried out with the LightShift® Chemoluinescent EMSA Kit (PIERCE) according to the product manual in final volume of 20 l. A 39 bp double stranded DNA substrate RUU172 (5′-TTAAATAATGATAATTTAAATTATCAAACAGTAACTTTT′) was generated by annealing two 5′-biotinylated ligonucleotides in 10 mM Tris/HCl pH 7.5, 100 mM NaCl and mM EDTA during a temperature gradient from 85 ◦C to 25 ◦C.

    Article Title: SNHG15-Mediated Localization of Nucleolin at the Cell Protrusions Regulates CDH2 mRNA Expression and Cell Invasion
    Article Snippet: .. Briefly, recombinant MBP–MCP-conjugated amylose resin (NEB, USA) was incubated with cell lysates prepared from cells expressing MS2-tagged full-length or truncated SNHG15 at 4 °C for 4 h in the presence of RNase and protease inhibitors. ..

    Article Title: p37/UBXN2B regulates spindle orientation by limiting cortical NuMA recruitment via PP1/Repo-Man
    Article Snippet: The full-length p37 cDNA was subcloned by Gateway technology (Invitrogen) into the pDEST–maltose-binding protein (MBP) vector. .. Recombinant MBP-tagged p37 (MBP-p37) protein expression was induced in BL21 Escherichia coli and purified using standard column purification on an amylose resin (New England Biolabs, Inc.) column. .. Purified MBP-p37 was used to immunize two rabbits with a standard immunization protocol (Moravian-Biotechnology).

    Article Title: Recombinant Vaccine Displaying the Loop-Neutralizing Determinant from Protective Antigen Completely Protects Rabbits from Experimental Inhalation Anthrax
    Article Snippet: All synthetic peptides were synthesized commercially (Sigma-Genosys, The Woodlands, TX) and were high-performance liquid chromatography (HPLC) purified to greater than 90% purity. .. Recombinant MBP (NEB, Carlsbad CA) was used as a test antigen in the lymph node proliferation assays. .. Approximately 12- to 16-week-old female C57BL/6, C3H, SJL, and BALB/c mice (Jackson Laboratory, Bar Harbor, ME) were used for the mouse experiments.

    Article Title: Human serine racemase structure/activity relationship studies provide mechanistic insight and point to position 84 as a hot spot for β-elimination function
    Article Snippet: .. Recombinant MBP–hSR The protein was designed to bear the N-terminal MBP tag present in the pMAL-c2X (New England Biolabs) vector. .. The following primers were employed in this system (IDT): 5′-CGT TGC GGA TCC ATG TGT GCT CAG TAT TGC-3′ (forward) and 5′-CAC CTA GTC GAC AAT TCC CAC CAT TTC C-3′ (reverse), for restriction sites BamHI and SalI, respectively.

    Article Title: Recombinant Vaccine Displaying the Loop-Neutralizing Determinant from Protective Antigen Completely Protects Rabbits from Experimental Inhalation Anthrax
    Article Snippet: All synthetic peptides were synthesized commercially (Sigma-Genosys, The Woodlands, TX) and were high-performance liquid chromatography (HPLC) purified to greater than 90% purity. .. Recombinant MBP (NEB, Carlsbad CA) was used as a test antigen in the lymph node proliferation assays. ..

    Article Title: Loss of ErbB3 redirects Integrin β1 from early endosomal recycling to secretion in extracellular vesicles
    Article Snippet: pMalC2–Rabaptin5 was transformed into the BL21S(DE3)pLysS bacterial strain (Invitrogen). .. Protein expression was induced with 0.3 mM IPTG for 2 h at 37°C, and recombinant MBP–Rabaptin5 was purified with an amylose resin (New England Biolabs) according to the manufacturer’s instructions and dialyzed against lysis buffer (20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 0.05 mM PMSF, 6 KIU7/ml aprotinin, 0.5 mM DTT, 5% glycerol, and 0.5% Triton X-100). .. Recombinant 6His-ErbB3 was purchased from Sigma-Aldrich.

    Incubation:

    Article Title: SNHG15-Mediated Localization of Nucleolin at the Cell Protrusions Regulates CDH2 mRNA Expression and Cell Invasion.
    Article Snippet: .. Briefly, recombinant MBP–MCP-conjugated amylose resin (NEB, USA) was incubated with cell lysates prepared from cells expressing MS2-tagged full-length or truncated SNHG15 at 4 ◦C for 4 h in the presence of RNase and protease inhibitors. ..

    Article Title: SNHG15-Mediated Localization of Nucleolin at the Cell Protrusions Regulates CDH2 mRNA Expression and Cell Invasion
    Article Snippet: .. Briefly, recombinant MBP–MCP-conjugated amylose resin (NEB, USA) was incubated with cell lysates prepared from cells expressing MS2-tagged full-length or truncated SNHG15 at 4 °C for 4 h in the presence of RNase and protease inhibitors. ..

    Expressing:

    Article Title: SNHG15-Mediated Localization of Nucleolin at the Cell Protrusions Regulates CDH2 mRNA Expression and Cell Invasion.
    Article Snippet: .. Briefly, recombinant MBP–MCP-conjugated amylose resin (NEB, USA) was incubated with cell lysates prepared from cells expressing MS2-tagged full-length or truncated SNHG15 at 4 ◦C for 4 h in the presence of RNase and protease inhibitors. ..

    Article Title: SNHG15-Mediated Localization of Nucleolin at the Cell Protrusions Regulates CDH2 mRNA Expression and Cell Invasion
    Article Snippet: .. Briefly, recombinant MBP–MCP-conjugated amylose resin (NEB, USA) was incubated with cell lysates prepared from cells expressing MS2-tagged full-length or truncated SNHG15 at 4 °C for 4 h in the presence of RNase and protease inhibitors. ..

    Article Title: p37/UBXN2B regulates spindle orientation by limiting cortical NuMA recruitment via PP1/Repo-Man
    Article Snippet: The full-length p37 cDNA was subcloned by Gateway technology (Invitrogen) into the pDEST–maltose-binding protein (MBP) vector. .. Recombinant MBP-tagged p37 (MBP-p37) protein expression was induced in BL21 Escherichia coli and purified using standard column purification on an amylose resin (New England Biolabs, Inc.) column. .. Purified MBP-p37 was used to immunize two rabbits with a standard immunization protocol (Moravian-Biotechnology).

    Article Title: Loss of ErbB3 redirects Integrin β1 from early endosomal recycling to secretion in extracellular vesicles
    Article Snippet: pMalC2–Rabaptin5 was transformed into the BL21S(DE3)pLysS bacterial strain (Invitrogen). .. Protein expression was induced with 0.3 mM IPTG for 2 h at 37°C, and recombinant MBP–Rabaptin5 was purified with an amylose resin (New England Biolabs) according to the manufacturer’s instructions and dialyzed against lysis buffer (20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 0.05 mM PMSF, 6 KIU7/ml aprotinin, 0.5 mM DTT, 5% glycerol, and 0.5% Triton X-100). .. Recombinant 6His-ErbB3 was purchased from Sigma-Aldrich.

    Transformation Assay:

    Article Title: XerC-mediated DNA inversion at the inverted repeats of the UU172-phase-variable element of Ureaplasma parvum serovar 3.
    Article Snippet: Zimmerman et al. / Microb vent was monitored by restriction analysis of the plasmids conaining the synthetic phase-variable element. aterials and methods ultivation and transformation of microorganisms E. coli Top10 F′ (F ́{lacIq Tn10 (TetR)} mcrA (mrr-hsdRMScrBC) 80lacZ M15 lacX74 recA1 araD139 (ara-leu)7697 alU galK rpsL endA1 nupG) (LifeTechnologies) was grown in broth r on agar containing 10 g L−1 trypton, 10 g L−1 yeast extract and g L−1 NaCl. .. Competent cells were transformed by heat shock at 3 ◦C for 2 min. urification of recombinant proteins The recombinant MBP (maltose binding protein) fusion proein MBP::XerC-6×His was purified as described (Zimmerman t al. 2013) by affinity chromatography over amylose resin accordng to the manufacturer’s protocol (NEB; http://www.neb.com/ ebecomm/manualfiles/manuale8000.pdf, Affinity Chromatograhy, Method I). .. The elution was then further purified over i2+-charged ProBondTM Resin (LifeTechnologies) and eluted with uffer (50 mM Tris, 300 mM NaCl, pH 7.5). lectrophoretic mobility shift assay (EMSA) EMSA analysis was carried out with the LightShift® Chemoluinescent EMSA Kit (PIERCE) according to the product manual in final volume of 20 l. A 39 bp double stranded DNA substrate RUU172 (5′-TTAAATAATGATAATTTAAATTATCAAACAGTAACTTTT′) was generated by annealing two 5′-biotinylated ligonucleotides in 10 mM Tris/HCl pH 7.5, 100 mM NaCl and mM EDTA during a temperature gradient from 85 ◦C to 25 ◦C.

    Binding Assay:

    Article Title: XerC-mediated DNA inversion at the inverted repeats of the UU172-phase-variable element of Ureaplasma parvum serovar 3.
    Article Snippet: Zimmerman et al. / Microb vent was monitored by restriction analysis of the plasmids conaining the synthetic phase-variable element. aterials and methods ultivation and transformation of microorganisms E. coli Top10 F′ (F ́{lacIq Tn10 (TetR)} mcrA (mrr-hsdRMScrBC) 80lacZ M15 lacX74 recA1 araD139 (ara-leu)7697 alU galK rpsL endA1 nupG) (LifeTechnologies) was grown in broth r on agar containing 10 g L−1 trypton, 10 g L−1 yeast extract and g L−1 NaCl. .. Competent cells were transformed by heat shock at 3 ◦C for 2 min. urification of recombinant proteins The recombinant MBP (maltose binding protein) fusion proein MBP::XerC-6×His was purified as described (Zimmerman t al. 2013) by affinity chromatography over amylose resin accordng to the manufacturer’s protocol (NEB; http://www.neb.com/ ebecomm/manualfiles/manuale8000.pdf, Affinity Chromatograhy, Method I). .. The elution was then further purified over i2+-charged ProBondTM Resin (LifeTechnologies) and eluted with uffer (50 mM Tris, 300 mM NaCl, pH 7.5). lectrophoretic mobility shift assay (EMSA) EMSA analysis was carried out with the LightShift® Chemoluinescent EMSA Kit (PIERCE) according to the product manual in final volume of 20 l. A 39 bp double stranded DNA substrate RUU172 (5′-TTAAATAATGATAATTTAAATTATCAAACAGTAACTTTT′) was generated by annealing two 5′-biotinylated ligonucleotides in 10 mM Tris/HCl pH 7.5, 100 mM NaCl and mM EDTA during a temperature gradient from 85 ◦C to 25 ◦C.

    Purification:

    Article Title: XerC-mediated DNA inversion at the inverted repeats of the UU172-phase-variable element of Ureaplasma parvum serovar 3.
    Article Snippet: Zimmerman et al. / Microb vent was monitored by restriction analysis of the plasmids conaining the synthetic phase-variable element. aterials and methods ultivation and transformation of microorganisms E. coli Top10 F′ (F ́{lacIq Tn10 (TetR)} mcrA (mrr-hsdRMScrBC) 80lacZ M15 lacX74 recA1 araD139 (ara-leu)7697 alU galK rpsL endA1 nupG) (LifeTechnologies) was grown in broth r on agar containing 10 g L−1 trypton, 10 g L−1 yeast extract and g L−1 NaCl. .. Competent cells were transformed by heat shock at 3 ◦C for 2 min. urification of recombinant proteins The recombinant MBP (maltose binding protein) fusion proein MBP::XerC-6×His was purified as described (Zimmerman t al. 2013) by affinity chromatography over amylose resin accordng to the manufacturer’s protocol (NEB; http://www.neb.com/ ebecomm/manualfiles/manuale8000.pdf, Affinity Chromatograhy, Method I). .. The elution was then further purified over i2+-charged ProBondTM Resin (LifeTechnologies) and eluted with uffer (50 mM Tris, 300 mM NaCl, pH 7.5). lectrophoretic mobility shift assay (EMSA) EMSA analysis was carried out with the LightShift® Chemoluinescent EMSA Kit (PIERCE) according to the product manual in final volume of 20 l. A 39 bp double stranded DNA substrate RUU172 (5′-TTAAATAATGATAATTTAAATTATCAAACAGTAACTTTT′) was generated by annealing two 5′-biotinylated ligonucleotides in 10 mM Tris/HCl pH 7.5, 100 mM NaCl and mM EDTA during a temperature gradient from 85 ◦C to 25 ◦C.

    Article Title: p37/UBXN2B regulates spindle orientation by limiting cortical NuMA recruitment via PP1/Repo-Man
    Article Snippet: The full-length p37 cDNA was subcloned by Gateway technology (Invitrogen) into the pDEST–maltose-binding protein (MBP) vector. .. Recombinant MBP-tagged p37 (MBP-p37) protein expression was induced in BL21 Escherichia coli and purified using standard column purification on an amylose resin (New England Biolabs, Inc.) column. .. Purified MBP-p37 was used to immunize two rabbits with a standard immunization protocol (Moravian-Biotechnology).

    Article Title: Loss of ErbB3 redirects Integrin β1 from early endosomal recycling to secretion in extracellular vesicles
    Article Snippet: pMalC2–Rabaptin5 was transformed into the BL21S(DE3)pLysS bacterial strain (Invitrogen). .. Protein expression was induced with 0.3 mM IPTG for 2 h at 37°C, and recombinant MBP–Rabaptin5 was purified with an amylose resin (New England Biolabs) according to the manufacturer’s instructions and dialyzed against lysis buffer (20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 0.05 mM PMSF, 6 KIU7/ml aprotinin, 0.5 mM DTT, 5% glycerol, and 0.5% Triton X-100). .. Recombinant 6His-ErbB3 was purchased from Sigma-Aldrich.

    Affinity Chromatography:

    Article Title: XerC-mediated DNA inversion at the inverted repeats of the UU172-phase-variable element of Ureaplasma parvum serovar 3.
    Article Snippet: Zimmerman et al. / Microb vent was monitored by restriction analysis of the plasmids conaining the synthetic phase-variable element. aterials and methods ultivation and transformation of microorganisms E. coli Top10 F′ (F ́{lacIq Tn10 (TetR)} mcrA (mrr-hsdRMScrBC) 80lacZ M15 lacX74 recA1 araD139 (ara-leu)7697 alU galK rpsL endA1 nupG) (LifeTechnologies) was grown in broth r on agar containing 10 g L−1 trypton, 10 g L−1 yeast extract and g L−1 NaCl. .. Competent cells were transformed by heat shock at 3 ◦C for 2 min. urification of recombinant proteins The recombinant MBP (maltose binding protein) fusion proein MBP::XerC-6×His was purified as described (Zimmerman t al. 2013) by affinity chromatography over amylose resin accordng to the manufacturer’s protocol (NEB; http://www.neb.com/ ebecomm/manualfiles/manuale8000.pdf, Affinity Chromatograhy, Method I). .. The elution was then further purified over i2+-charged ProBondTM Resin (LifeTechnologies) and eluted with uffer (50 mM Tris, 300 mM NaCl, pH 7.5). lectrophoretic mobility shift assay (EMSA) EMSA analysis was carried out with the LightShift® Chemoluinescent EMSA Kit (PIERCE) according to the product manual in final volume of 20 l. A 39 bp double stranded DNA substrate RUU172 (5′-TTAAATAATGATAATTTAAATTATCAAACAGTAACTTTT′) was generated by annealing two 5′-biotinylated ligonucleotides in 10 mM Tris/HCl pH 7.5, 100 mM NaCl and mM EDTA during a temperature gradient from 85 ◦C to 25 ◦C.

    Lysis:

    Article Title: Loss of ErbB3 redirects Integrin β1 from early endosomal recycling to secretion in extracellular vesicles
    Article Snippet: pMalC2–Rabaptin5 was transformed into the BL21S(DE3)pLysS bacterial strain (Invitrogen). .. Protein expression was induced with 0.3 mM IPTG for 2 h at 37°C, and recombinant MBP–Rabaptin5 was purified with an amylose resin (New England Biolabs) according to the manufacturer’s instructions and dialyzed against lysis buffer (20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 0.05 mM PMSF, 6 KIU7/ml aprotinin, 0.5 mM DTT, 5% glycerol, and 0.5% Triton X-100). .. Recombinant 6His-ErbB3 was purchased from Sigma-Aldrich.



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    Eosinophil-deficient 129 ΔdblGATA1 mice and 129 WT mice were orally infected with tdTomato-expressing S Tm (10 8 c.f.u.s) for 4 weeks to assess eosinophil-dependent bacterial control in the MLN. a , Representative FACS plots of tdTomato- S Tm in 129 WT and 129 ΔdblGATA1 mice at 4 w.p.i. b , Frequency of tdTomato- S Tm + immune cells in 129 WT and 129 ΔdblGATA1 mice ( n = 7–8 mice per group, 2 experiments). c , Representative FACS histogram of tdTomato- S Tm + macrophages (Mϕ) in 129 WT (orange) and 129 ΔdblGATA1 (grey). Uninfected controls (black) served as a negative control. d , MFI of tdTomato- S Tm within Mϕ in 129 WT and 129 ΔdblGATA1 mice. e , Frequency of tdTomato- S Tm + iNOS + or tdTomato- S Tm + CD206 + Mϕ in 129 WT and 129 ΔdblGATA1 mice ( n = 7–8 mice per group, 2 experiments). f , MFI of tdTomato- S Tm within iNOS + , CD206 + and iNOS − CD206 − Mϕ in 129 WT and 129 ΔdblGATA1 mice ( n = 7–8 mice per group, 2 experiments) (see also Extended Data Fig. ). g – i , Western blots of iNOS and GAPDH in whole-cell lysates from 129 WT BMDMs stimulated with <t>recombinant</t> EPX (100 ng ml −1 ), <t>MBP</t> (100 ng ml −1 ), MBP and EPX (100 ng ml −1 each), LPS (100 ng ml −1 ) or IL-4 (20 ng ml −1 ) ( g ); dose-dependent MBP effects (1–100 ng ml −1 ) ( h ); kinetics of MBP effects (10 ng ml −1 , 1–24 h) ( i ). n = 3 biological replicates (see also Extended Data Fig. ). j , Intracellular ROS levels measured over time in 129 WT BMDMs stimulated with MBP (10 ng ml −1 ) and LPS (100 ng ml −1 ) ( n = 3 biological replicates). k , Western blots of iNOS and GAPDH in 129 WT BMDMs stimulated with MBP, heat-inactivated (HI)-MBP, LPS or HI-LPS for 24 h (3 biological replicates). l , Fold-replication of S Tm in 129 WT BMDMs pre-stimulated with MBP (10 ng ml −1 ), EPX (10 ng ml −1 ), LPS (100 ng ml −1 ) or IL-4 (20 ng ml −1 ) for 24 h before gentamycin protection assay (bacteria enumerated at 2 h and 24 h, 4 biological replicates). m – p , Schematic of MBP neutralization using anti-MBP antibodies. Mice were infected with S Tm for 4 weeks and then given 5 daily doses of anti-IgG 2 or anti-MBP antibodies before sacrifice on day 33 ( m ); western blots of iNOS, MBP and GAPDH in whole MLN lysates from uninfected and infected mice treated with anti-IgG 2 or anti-MBP at 33 days post infection ( n ); quantification of iNOS (left) and MBP (right) protein levels in MBP-neutralized mice compared to IgG 2 -treated mice and uninfected controls ( n = 3–9 mice per group, 2 experiments) ( o ); MLN bacterial burden in MBP-neutralized mice compared to IgG2-treated mice ( n = 9 mice per group, 2 experiments) ( p ). q , Proposed model showing how macrophages and monocytes recruit eosinophils to the MLN through CCL11-dependent signalling. Recruited eosinophils become activated in S Tm-infected MLN and release MBP which leads to S Tm control within macrophages, increased macrophage iNOS levels and sustained granuloma integrity. All analyses are two-tailed and data were analysed using Mann–Whitney U -test ( b , d , e , p ); two-way ANOVA with uncorrected Fisher’s LSD test ( f ), two-way ANOVA with Tukey correction ( j ), one-way ANOVA with Tukey correction ( l ) or Kruskal–Wallis with Dunn’s post hoc test ( o ). Exact P values in Supplementary Table . * P < 0.05, ** P < 0.01, *** P < 0.001.
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    Eosinophil-deficient 129 ΔdblGATA1 mice and 129 WT mice were orally infected with tdTomato-expressing S Tm (10 8 c.f.u.s) for 4 weeks to assess eosinophil-dependent bacterial control in the MLN. a , Representative FACS plots of tdTomato- S Tm in 129 WT and 129 ΔdblGATA1 mice at 4 w.p.i. b , Frequency of tdTomato- S Tm + immune cells in 129 WT and 129 ΔdblGATA1 mice ( n = 7–8 mice per group, 2 experiments). c , Representative FACS histogram of tdTomato- S Tm + macrophages (Mϕ) in 129 WT (orange) and 129 ΔdblGATA1 (grey). Uninfected controls (black) served as a negative control. d , MFI of tdTomato- S Tm within Mϕ in 129 WT and 129 ΔdblGATA1 mice. e , Frequency of tdTomato- S Tm + iNOS + or tdTomato- S Tm + CD206 + Mϕ in 129 WT and 129 ΔdblGATA1 mice ( n = 7–8 mice per group, 2 experiments). f , MFI of tdTomato- S Tm within iNOS + , CD206 + and iNOS − CD206 − Mϕ in 129 WT and 129 ΔdblGATA1 mice ( n = 7–8 mice per group, 2 experiments) (see also Extended Data Fig. ). g – i , Western blots of iNOS and GAPDH in whole-cell lysates from 129 WT BMDMs stimulated with <t>recombinant</t> EPX (100 ng ml −1 ), <t>MBP</t> (100 ng ml −1 ), MBP and EPX (100 ng ml −1 each), LPS (100 ng ml −1 ) or IL-4 (20 ng ml −1 ) ( g ); dose-dependent MBP effects (1–100 ng ml −1 ) ( h ); kinetics of MBP effects (10 ng ml −1 , 1–24 h) ( i ). n = 3 biological replicates (see also Extended Data Fig. ). j , Intracellular ROS levels measured over time in 129 WT BMDMs stimulated with MBP (10 ng ml −1 ) and LPS (100 ng ml −1 ) ( n = 3 biological replicates). k , Western blots of iNOS and GAPDH in 129 WT BMDMs stimulated with MBP, heat-inactivated (HI)-MBP, LPS or HI-LPS for 24 h (3 biological replicates). l , Fold-replication of S Tm in 129 WT BMDMs pre-stimulated with MBP (10 ng ml −1 ), EPX (10 ng ml −1 ), LPS (100 ng ml −1 ) or IL-4 (20 ng ml −1 ) for 24 h before gentamycin protection assay (bacteria enumerated at 2 h and 24 h, 4 biological replicates). m – p , Schematic of MBP neutralization using anti-MBP antibodies. Mice were infected with S Tm for 4 weeks and then given 5 daily doses of anti-IgG 2 or anti-MBP antibodies before sacrifice on day 33 ( m ); western blots of iNOS, MBP and GAPDH in whole MLN lysates from uninfected and infected mice treated with anti-IgG 2 or anti-MBP at 33 days post infection ( n ); quantification of iNOS (left) and MBP (right) protein levels in MBP-neutralized mice compared to IgG 2 -treated mice and uninfected controls ( n = 3–9 mice per group, 2 experiments) ( o ); MLN bacterial burden in MBP-neutralized mice compared to IgG2-treated mice ( n = 9 mice per group, 2 experiments) ( p ). q , Proposed model showing how macrophages and monocytes recruit eosinophils to the MLN through CCL11-dependent signalling. Recruited eosinophils become activated in S Tm-infected MLN and release MBP which leads to S Tm control within macrophages, increased macrophage iNOS levels and sustained granuloma integrity. All analyses are two-tailed and data were analysed using Mann–Whitney U -test ( b , d , e , p ); two-way ANOVA with uncorrected Fisher’s LSD test ( f ), two-way ANOVA with Tukey correction ( j ), one-way ANOVA with Tukey correction ( l ) or Kruskal–Wallis with Dunn’s post hoc test ( o ). Exact P values in Supplementary Table . * P < 0.05, ** P < 0.01, *** P < 0.001.
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    Addgene inc n a recombinant dna pj4m tdp 43 tev mbp his6 addgene 104480 png2 vector barghorn et
    Eosinophil-deficient 129 ΔdblGATA1 mice and 129 WT mice were orally infected with tdTomato-expressing S Tm (10 8 c.f.u.s) for 4 weeks to assess eosinophil-dependent bacterial control in the MLN. a , Representative FACS plots of tdTomato- S Tm in 129 WT and 129 ΔdblGATA1 mice at 4 w.p.i. b , Frequency of tdTomato- S Tm + immune cells in 129 WT and 129 ΔdblGATA1 mice ( n = 7–8 mice per group, 2 experiments). c , Representative FACS histogram of tdTomato- S Tm + macrophages (Mϕ) in 129 WT (orange) and 129 ΔdblGATA1 (grey). Uninfected controls (black) served as a negative control. d , MFI of tdTomato- S Tm within Mϕ in 129 WT and 129 ΔdblGATA1 mice. e , Frequency of tdTomato- S Tm + iNOS + or tdTomato- S Tm + CD206 + Mϕ in 129 WT and 129 ΔdblGATA1 mice ( n = 7–8 mice per group, 2 experiments). f , MFI of tdTomato- S Tm within iNOS + , CD206 + and iNOS − CD206 − Mϕ in 129 WT and 129 ΔdblGATA1 mice ( n = 7–8 mice per group, 2 experiments) (see also Extended Data Fig. ). g – i , Western blots of iNOS and GAPDH in whole-cell lysates from 129 WT BMDMs stimulated with <t>recombinant</t> EPX (100 ng ml −1 ), <t>MBP</t> (100 ng ml −1 ), MBP and EPX (100 ng ml −1 each), LPS (100 ng ml −1 ) or IL-4 (20 ng ml −1 ) ( g ); dose-dependent MBP effects (1–100 ng ml −1 ) ( h ); kinetics of MBP effects (10 ng ml −1 , 1–24 h) ( i ). n = 3 biological replicates (see also Extended Data Fig. ). j , Intracellular ROS levels measured over time in 129 WT BMDMs stimulated with MBP (10 ng ml −1 ) and LPS (100 ng ml −1 ) ( n = 3 biological replicates). k , Western blots of iNOS and GAPDH in 129 WT BMDMs stimulated with MBP, heat-inactivated (HI)-MBP, LPS or HI-LPS for 24 h (3 biological replicates). l , Fold-replication of S Tm in 129 WT BMDMs pre-stimulated with MBP (10 ng ml −1 ), EPX (10 ng ml −1 ), LPS (100 ng ml −1 ) or IL-4 (20 ng ml −1 ) for 24 h before gentamycin protection assay (bacteria enumerated at 2 h and 24 h, 4 biological replicates). m – p , Schematic of MBP neutralization using anti-MBP antibodies. Mice were infected with S Tm for 4 weeks and then given 5 daily doses of anti-IgG 2 or anti-MBP antibodies before sacrifice on day 33 ( m ); western blots of iNOS, MBP and GAPDH in whole MLN lysates from uninfected and infected mice treated with anti-IgG 2 or anti-MBP at 33 days post infection ( n ); quantification of iNOS (left) and MBP (right) protein levels in MBP-neutralized mice compared to IgG 2 -treated mice and uninfected controls ( n = 3–9 mice per group, 2 experiments) ( o ); MLN bacterial burden in MBP-neutralized mice compared to IgG2-treated mice ( n = 9 mice per group, 2 experiments) ( p ). q , Proposed model showing how macrophages and monocytes recruit eosinophils to the MLN through CCL11-dependent signalling. Recruited eosinophils become activated in S Tm-infected MLN and release MBP which leads to S Tm control within macrophages, increased macrophage iNOS levels and sustained granuloma integrity. All analyses are two-tailed and data were analysed using Mann–Whitney U -test ( b , d , e , p ); two-way ANOVA with uncorrected Fisher’s LSD test ( f ), two-way ANOVA with Tukey correction ( j ), one-way ANOVA with Tukey correction ( l ) or Kruskal–Wallis with Dunn’s post hoc test ( o ). Exact P values in Supplementary Table . * P < 0.05, ** P < 0.01, *** P < 0.001.
    N A Recombinant Dna Pj4m Tdp 43 Tev Mbp His6 Addgene 104480 Png2 Vector Barghorn Et, supplied by Addgene inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Eosinophil-deficient 129 ΔdblGATA1 mice and 129 WT mice were orally infected with tdTomato-expressing S Tm (10 8 c.f.u.s) for 4 weeks to assess eosinophil-dependent bacterial control in the MLN. a , Representative FACS plots of tdTomato- S Tm in 129 WT and 129 ΔdblGATA1 mice at 4 w.p.i. b , Frequency of tdTomato- S Tm + immune cells in 129 WT and 129 ΔdblGATA1 mice ( n = 7–8 mice per group, 2 experiments). c , Representative FACS histogram of tdTomato- S Tm + macrophages (Mϕ) in 129 WT (orange) and 129 ΔdblGATA1 (grey). Uninfected controls (black) served as a negative control. d , MFI of tdTomato- S Tm within Mϕ in 129 WT and 129 ΔdblGATA1 mice. e , Frequency of tdTomato- S Tm + iNOS + or tdTomato- S Tm + CD206 + Mϕ in 129 WT and 129 ΔdblGATA1 mice ( n = 7–8 mice per group, 2 experiments). f , MFI of tdTomato- S Tm within iNOS + , CD206 + and iNOS − CD206 − Mϕ in 129 WT and 129 ΔdblGATA1 mice ( n = 7–8 mice per group, 2 experiments) (see also Extended Data Fig. ). g – i , Western blots of iNOS and GAPDH in whole-cell lysates from 129 WT BMDMs stimulated with <t>recombinant</t> EPX (100 ng ml −1 ), <t>MBP</t> (100 ng ml −1 ), MBP and EPX (100 ng ml −1 each), LPS (100 ng ml −1 ) or IL-4 (20 ng ml −1 ) ( g ); dose-dependent MBP effects (1–100 ng ml −1 ) ( h ); kinetics of MBP effects (10 ng ml −1 , 1–24 h) ( i ). n = 3 biological replicates (see also Extended Data Fig. ). j , Intracellular ROS levels measured over time in 129 WT BMDMs stimulated with MBP (10 ng ml −1 ) and LPS (100 ng ml −1 ) ( n = 3 biological replicates). k , Western blots of iNOS and GAPDH in 129 WT BMDMs stimulated with MBP, heat-inactivated (HI)-MBP, LPS or HI-LPS for 24 h (3 biological replicates). l , Fold-replication of S Tm in 129 WT BMDMs pre-stimulated with MBP (10 ng ml −1 ), EPX (10 ng ml −1 ), LPS (100 ng ml −1 ) or IL-4 (20 ng ml −1 ) for 24 h before gentamycin protection assay (bacteria enumerated at 2 h and 24 h, 4 biological replicates). m – p , Schematic of MBP neutralization using anti-MBP antibodies. Mice were infected with S Tm for 4 weeks and then given 5 daily doses of anti-IgG 2 or anti-MBP antibodies before sacrifice on day 33 ( m ); western blots of iNOS, MBP and GAPDH in whole MLN lysates from uninfected and infected mice treated with anti-IgG 2 or anti-MBP at 33 days post infection ( n ); quantification of iNOS (left) and MBP (right) protein levels in MBP-neutralized mice compared to IgG 2 -treated mice and uninfected controls ( n = 3–9 mice per group, 2 experiments) ( o ); MLN bacterial burden in MBP-neutralized mice compared to IgG2-treated mice ( n = 9 mice per group, 2 experiments) ( p ). q , Proposed model showing how macrophages and monocytes recruit eosinophils to the MLN through CCL11-dependent signalling. Recruited eosinophils become activated in S Tm-infected MLN and release MBP which leads to S Tm control within macrophages, increased macrophage iNOS levels and sustained granuloma integrity. All analyses are two-tailed and data were analysed using Mann–Whitney U -test ( b , d , e , p ); two-way ANOVA with uncorrected Fisher’s LSD test ( f ), two-way ANOVA with Tukey correction ( j ), one-way ANOVA with Tukey correction ( l ) or Kruskal–Wallis with Dunn’s post hoc test ( o ). Exact P values in Supplementary Table . * P < 0.05, ** P < 0.01, *** P < 0.001.
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    Eosinophil-deficient 129 ΔdblGATA1 mice and 129 WT mice were orally infected with tdTomato-expressing S Tm (10 8 c.f.u.s) for 4 weeks to assess eosinophil-dependent bacterial control in the MLN. a , Representative FACS plots of tdTomato- S Tm in 129 WT and 129 ΔdblGATA1 mice at 4 w.p.i. b , Frequency of tdTomato- S Tm + immune cells in 129 WT and 129 ΔdblGATA1 mice ( n = 7–8 mice per group, 2 experiments). c , Representative FACS histogram of tdTomato- S Tm + macrophages (Mϕ) in 129 WT (orange) and 129 ΔdblGATA1 (grey). Uninfected controls (black) served as a negative control. d , MFI of tdTomato- S Tm within Mϕ in 129 WT and 129 ΔdblGATA1 mice. e , Frequency of tdTomato- S Tm + iNOS + or tdTomato- S Tm + CD206 + Mϕ in 129 WT and 129 ΔdblGATA1 mice ( n = 7–8 mice per group, 2 experiments). f , MFI of tdTomato- S Tm within iNOS + , CD206 + and iNOS − CD206 − Mϕ in 129 WT and 129 ΔdblGATA1 mice ( n = 7–8 mice per group, 2 experiments) (see also Extended Data Fig. ). g – i , Western blots of iNOS and GAPDH in whole-cell lysates from 129 WT BMDMs stimulated with recombinant EPX (100 ng ml −1 ), MBP (100 ng ml −1 ), MBP and EPX (100 ng ml −1 each), LPS (100 ng ml −1 ) or IL-4 (20 ng ml −1 ) ( g ); dose-dependent MBP effects (1–100 ng ml −1 ) ( h ); kinetics of MBP effects (10 ng ml −1 , 1–24 h) ( i ). n = 3 biological replicates (see also Extended Data Fig. ). j , Intracellular ROS levels measured over time in 129 WT BMDMs stimulated with MBP (10 ng ml −1 ) and LPS (100 ng ml −1 ) ( n = 3 biological replicates). k , Western blots of iNOS and GAPDH in 129 WT BMDMs stimulated with MBP, heat-inactivated (HI)-MBP, LPS or HI-LPS for 24 h (3 biological replicates). l , Fold-replication of S Tm in 129 WT BMDMs pre-stimulated with MBP (10 ng ml −1 ), EPX (10 ng ml −1 ), LPS (100 ng ml −1 ) or IL-4 (20 ng ml −1 ) for 24 h before gentamycin protection assay (bacteria enumerated at 2 h and 24 h, 4 biological replicates). m – p , Schematic of MBP neutralization using anti-MBP antibodies. Mice were infected with S Tm for 4 weeks and then given 5 daily doses of anti-IgG 2 or anti-MBP antibodies before sacrifice on day 33 ( m ); western blots of iNOS, MBP and GAPDH in whole MLN lysates from uninfected and infected mice treated with anti-IgG 2 or anti-MBP at 33 days post infection ( n ); quantification of iNOS (left) and MBP (right) protein levels in MBP-neutralized mice compared to IgG 2 -treated mice and uninfected controls ( n = 3–9 mice per group, 2 experiments) ( o ); MLN bacterial burden in MBP-neutralized mice compared to IgG2-treated mice ( n = 9 mice per group, 2 experiments) ( p ). q , Proposed model showing how macrophages and monocytes recruit eosinophils to the MLN through CCL11-dependent signalling. Recruited eosinophils become activated in S Tm-infected MLN and release MBP which leads to S Tm control within macrophages, increased macrophage iNOS levels and sustained granuloma integrity. All analyses are two-tailed and data were analysed using Mann–Whitney U -test ( b , d , e , p ); two-way ANOVA with uncorrected Fisher’s LSD test ( f ), two-way ANOVA with Tukey correction ( j ), one-way ANOVA with Tukey correction ( l ) or Kruskal–Wallis with Dunn’s post hoc test ( o ). Exact P values in Supplementary Table . * P < 0.05, ** P < 0.01, *** P < 0.001.

    Journal: Nature Microbiology

    Article Title: Eosinophils enhance granuloma-mediated control of persistent Salmonella infection in mice

    doi: 10.1038/s41564-025-02187-1

    Figure Lengend Snippet: Eosinophil-deficient 129 ΔdblGATA1 mice and 129 WT mice were orally infected with tdTomato-expressing S Tm (10 8 c.f.u.s) for 4 weeks to assess eosinophil-dependent bacterial control in the MLN. a , Representative FACS plots of tdTomato- S Tm in 129 WT and 129 ΔdblGATA1 mice at 4 w.p.i. b , Frequency of tdTomato- S Tm + immune cells in 129 WT and 129 ΔdblGATA1 mice ( n = 7–8 mice per group, 2 experiments). c , Representative FACS histogram of tdTomato- S Tm + macrophages (Mϕ) in 129 WT (orange) and 129 ΔdblGATA1 (grey). Uninfected controls (black) served as a negative control. d , MFI of tdTomato- S Tm within Mϕ in 129 WT and 129 ΔdblGATA1 mice. e , Frequency of tdTomato- S Tm + iNOS + or tdTomato- S Tm + CD206 + Mϕ in 129 WT and 129 ΔdblGATA1 mice ( n = 7–8 mice per group, 2 experiments). f , MFI of tdTomato- S Tm within iNOS + , CD206 + and iNOS − CD206 − Mϕ in 129 WT and 129 ΔdblGATA1 mice ( n = 7–8 mice per group, 2 experiments) (see also Extended Data Fig. ). g – i , Western blots of iNOS and GAPDH in whole-cell lysates from 129 WT BMDMs stimulated with recombinant EPX (100 ng ml −1 ), MBP (100 ng ml −1 ), MBP and EPX (100 ng ml −1 each), LPS (100 ng ml −1 ) or IL-4 (20 ng ml −1 ) ( g ); dose-dependent MBP effects (1–100 ng ml −1 ) ( h ); kinetics of MBP effects (10 ng ml −1 , 1–24 h) ( i ). n = 3 biological replicates (see also Extended Data Fig. ). j , Intracellular ROS levels measured over time in 129 WT BMDMs stimulated with MBP (10 ng ml −1 ) and LPS (100 ng ml −1 ) ( n = 3 biological replicates). k , Western blots of iNOS and GAPDH in 129 WT BMDMs stimulated with MBP, heat-inactivated (HI)-MBP, LPS or HI-LPS for 24 h (3 biological replicates). l , Fold-replication of S Tm in 129 WT BMDMs pre-stimulated with MBP (10 ng ml −1 ), EPX (10 ng ml −1 ), LPS (100 ng ml −1 ) or IL-4 (20 ng ml −1 ) for 24 h before gentamycin protection assay (bacteria enumerated at 2 h and 24 h, 4 biological replicates). m – p , Schematic of MBP neutralization using anti-MBP antibodies. Mice were infected with S Tm for 4 weeks and then given 5 daily doses of anti-IgG 2 or anti-MBP antibodies before sacrifice on day 33 ( m ); western blots of iNOS, MBP and GAPDH in whole MLN lysates from uninfected and infected mice treated with anti-IgG 2 or anti-MBP at 33 days post infection ( n ); quantification of iNOS (left) and MBP (right) protein levels in MBP-neutralized mice compared to IgG 2 -treated mice and uninfected controls ( n = 3–9 mice per group, 2 experiments) ( o ); MLN bacterial burden in MBP-neutralized mice compared to IgG2-treated mice ( n = 9 mice per group, 2 experiments) ( p ). q , Proposed model showing how macrophages and monocytes recruit eosinophils to the MLN through CCL11-dependent signalling. Recruited eosinophils become activated in S Tm-infected MLN and release MBP which leads to S Tm control within macrophages, increased macrophage iNOS levels and sustained granuloma integrity. All analyses are two-tailed and data were analysed using Mann–Whitney U -test ( b , d , e , p ); two-way ANOVA with uncorrected Fisher’s LSD test ( f ), two-way ANOVA with Tukey correction ( j ), one-way ANOVA with Tukey correction ( l ) or Kruskal–Wallis with Dunn’s post hoc test ( o ). Exact P values in Supplementary Table . * P < 0.05, ** P < 0.01, *** P < 0.001.

    Article Snippet: In vitro polarization was performed using recombinant mouse MBP or EPX expressed in E. coli (Abbexa abx653285 and Abbexa abx067910) or commercial recombinant human MBP expressed in HEK-293 cells (Elabscience, PKSH032124).

    Techniques: Infection, Expressing, Control, Negative Control, Western Blot, Recombinant, Bacteria, Neutralization, Two Tailed Test, MANN-WHITNEY

    ( a ) Representative immunofluorescence image of MLN tissue sections from mice at 4 w.p.i. showing CD11b (magenta), iNOS (Grey), EPX (Blue) and MBP (Green). Scale bar = 50 μm ( n = 5 mice, 2 sections each). ( b ) S Tm growth in media containing either PBS (Ctrl), EPX (0.155-1.55 μg/ml) or MBP (90-900 ng/ml) for 18 h (n = 3 biological repeats). ( c ) Representative FACS plots showing iNOS and Arginase-1 in MBP (100 ng/ml), EPX (100 ng/ml) MBP and EPX (100 ng/ml each), LPS (100 ng/ml) or IL-4 (20 ng/ml) stimulated BMDMs from 129×1/SvJ mice. ( d ) Quantification of percentage of iNOS+ and Arginase-1+ cells in MBP (100 ng/ml), EPX (100 ng/ml) MPB and EPX (100 ng/ml each), LPS (100 ng/ml) and IL-4 (20 ng/ml) stimulated BMDMs from 129×1/SvJ mice (n = BMDMs from 5 mice across two experiments). ( e ) Quantification of iNOS levels by western blots in main Fig. (n = 3 biological repeats). ( f ) Representative western blot showing iNOS expression in murine (mMBP), human (hMBP) (10 ng/ml each) or LPS treated BMDMs from 129×1/SvJ mice (n = 2 biological repeats). ( g ) Quantification of cell death as determined by SYTOX green positive cells (n = 3 biological repeats) ( h ) Frequencies of eosinophils, neutrophils, mast cells and, basophils in the MLN of 129×1/SvJ mice. Basophils are gated as Live, CD45 + CD200R3 + c-Kit − . Mast cells are gated as Live, CD45 + CD200R3 + c-Kit + . Data are presented as Means ± SEM. All analyses are two-tailed and analyzed by Kruskal Wallis with uncorrected Dunn’s test with Dunn’s correction ( b ) or one-way ANOVA ( d , e ). Exact P- values in Supplementary Table . ns = non-significant, * P < 0.05, ** P < 0.01. *** P < 0.001.

    Journal: Nature Microbiology

    Article Title: Eosinophils enhance granuloma-mediated control of persistent Salmonella infection in mice

    doi: 10.1038/s41564-025-02187-1

    Figure Lengend Snippet: ( a ) Representative immunofluorescence image of MLN tissue sections from mice at 4 w.p.i. showing CD11b (magenta), iNOS (Grey), EPX (Blue) and MBP (Green). Scale bar = 50 μm ( n = 5 mice, 2 sections each). ( b ) S Tm growth in media containing either PBS (Ctrl), EPX (0.155-1.55 μg/ml) or MBP (90-900 ng/ml) for 18 h (n = 3 biological repeats). ( c ) Representative FACS plots showing iNOS and Arginase-1 in MBP (100 ng/ml), EPX (100 ng/ml) MBP and EPX (100 ng/ml each), LPS (100 ng/ml) or IL-4 (20 ng/ml) stimulated BMDMs from 129×1/SvJ mice. ( d ) Quantification of percentage of iNOS+ and Arginase-1+ cells in MBP (100 ng/ml), EPX (100 ng/ml) MPB and EPX (100 ng/ml each), LPS (100 ng/ml) and IL-4 (20 ng/ml) stimulated BMDMs from 129×1/SvJ mice (n = BMDMs from 5 mice across two experiments). ( e ) Quantification of iNOS levels by western blots in main Fig. (n = 3 biological repeats). ( f ) Representative western blot showing iNOS expression in murine (mMBP), human (hMBP) (10 ng/ml each) or LPS treated BMDMs from 129×1/SvJ mice (n = 2 biological repeats). ( g ) Quantification of cell death as determined by SYTOX green positive cells (n = 3 biological repeats) ( h ) Frequencies of eosinophils, neutrophils, mast cells and, basophils in the MLN of 129×1/SvJ mice. Basophils are gated as Live, CD45 + CD200R3 + c-Kit − . Mast cells are gated as Live, CD45 + CD200R3 + c-Kit + . Data are presented as Means ± SEM. All analyses are two-tailed and analyzed by Kruskal Wallis with uncorrected Dunn’s test with Dunn’s correction ( b ) or one-way ANOVA ( d , e ). Exact P- values in Supplementary Table . ns = non-significant, * P < 0.05, ** P < 0.01. *** P < 0.001.

    Article Snippet: In vitro polarization was performed using recombinant mouse MBP or EPX expressed in E. coli (Abbexa abx653285 and Abbexa abx067910) or commercial recombinant human MBP expressed in HEK-293 cells (Elabscience, PKSH032124).

    Techniques: Immunofluorescence, Western Blot, Expressing, Two Tailed Test