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  • 99
    Millipore western blot wb
    Western Blot Wb, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad western blot wb
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    Santa Cruz Biotechnology western immunoblotting wb
    Western Immunoblotting Wb, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc western blotting wb
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    Applygen Technologies western blot wb kits
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    GE Healthcare western immunoblotting
    Comparison of secretion capacity in the HAP-less and cap-less strains. E. coli MC1000 Δ fliC Δ flgKL (ΔCKL) and Δ fliC D containing the plasmid pTrc-FliC-ΔD3 were grown in LB supplemented with 0.05 mM IPTG, and harvested at OD 600 1.5. Samples which represented either 25 µL or 400 µL culture media were loaded for SDS-PAGE, for intracellular and secreted protein respectively. a Representative images showing the secreted fractions following Coomassie staining, and the intracellular fractions following <t>immunoblotting</t> using an anti-flagellin (H48) antibody and a HRP secondary. A FliC-ΔD3 protein standard (S) was also included, to allow quantification of protein concentration by densitometry ( b , c ). Quantifications for biological triplicates, ± SE and individual data points shown, *p
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    NanoSight western blotting
    Comparison of secretion capacity in the HAP-less and cap-less strains. E. coli MC1000 Δ fliC Δ flgKL (ΔCKL) and Δ fliC D containing the plasmid pTrc-FliC-ΔD3 were grown in LB supplemented with 0.05 mM IPTG, and harvested at OD 600 1.5. Samples which represented either 25 µL or 400 µL culture media were loaded for SDS-PAGE, for intracellular and secreted protein respectively. a Representative images showing the secreted fractions following Coomassie staining, and the intracellular fractions following <t>immunoblotting</t> using an anti-flagellin (H48) antibody and a HRP secondary. A FliC-ΔD3 protein standard (S) was also included, to allow quantification of protein concentration by densitometry ( b , c ). Quantifications for biological triplicates, ± SE and individual data points shown, *p
    Western Blotting, supplied by NanoSight, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad western immunoblotting
    Analysis of Obg expression in M . synoviae strains using SDS-PAGE and <t>immunoblotting.</t> (A) SDS-PAGE (7.5%) of whole cell proteins from M . synoviae MS-H transformed with pMAS-LoriC (clone MS-H-C28) and pKS-VOTL (clones MS-H-T75, MS-H-T78 and MS-H-T90) plasmids, stained with Coomassie brilliant blue R-250 (Bio-Rad). Location of ~ 50 kDa protein in pKS-VOTL transformants is indicated with an arrow. (B) Immunostaining of untransformed 86079/7NS, MS-H, and transformed MS-H with pMAS-LoriC (clone MS-H-C28) and pKS-VOTL (clones MS-H-T75, MS-H-T78 and MS-H-T90) using polyclonal rabbit serum against M . synoviae Obg_3 peptide. Location of Obg-MBP fusion protein and overexpressed Obg is indicated with arrows on the right. (C) SDS-PAGE (8.75%) of whole cell proteins from M . synoviae 86079/7NS transformed with pMAS-LoriC (clone 7NS-C12 and 7NS-C38) and pKS-VOTL (clones 7NS-T30, 7NS-T20, 7NS-T8 and 7NS-T4) plasmids, stained with Coomassie brilliant blue R-250 (Bio-Rad). (D) Immunostaining of untransformed 86079/7NS and transformed 86079/7NS with pMAS-LoriC (clone 7NS-C12 and 7NS-C38) and pKS-VOTL (clones 7NS-T30, 7NS-T20, 7NS-8 and 7NS-T4) using polyclonal rabbit serum against M . synoviae Obg_1 peptide. Location of overexpressed Obg is indicated with an arrow on the right. (E) For comparison of Obg expression in MS-H and 86079/7NS, approximately equal amounts (88 μg/lane) of whole cell proteins from MS-H, MS-H-C28, MS-H-T75, 86079/7NS, 7NS-C38 and 7NS-T30 were separated by SDS-PAGE (8.75%) and stained with Coomassie Brilliant Blue. (F) Immunostaining at two different concentrations of whole cell proteins (i.e. 1/3 and 2/3 of that loaded onto SDS-PAGE as shown in panel E) of each strain/transformant using polyclonal rabbit serum against M . synoviae Obg_1 peptide. MW, protein marker (Precision Plus Protein, Dual Color, Bio-Rad).
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    Santa Cruz Biotechnology western blotting
    Analysis of Obg expression in M . synoviae strains using SDS-PAGE and <t>immunoblotting.</t> (A) SDS-PAGE (7.5%) of whole cell proteins from M . synoviae MS-H transformed with pMAS-LoriC (clone MS-H-C28) and pKS-VOTL (clones MS-H-T75, MS-H-T78 and MS-H-T90) plasmids, stained with Coomassie brilliant blue R-250 (Bio-Rad). Location of ~ 50 kDa protein in pKS-VOTL transformants is indicated with an arrow. (B) Immunostaining of untransformed 86079/7NS, MS-H, and transformed MS-H with pMAS-LoriC (clone MS-H-C28) and pKS-VOTL (clones MS-H-T75, MS-H-T78 and MS-H-T90) using polyclonal rabbit serum against M . synoviae Obg_3 peptide. Location of Obg-MBP fusion protein and overexpressed Obg is indicated with arrows on the right. (C) SDS-PAGE (8.75%) of whole cell proteins from M . synoviae 86079/7NS transformed with pMAS-LoriC (clone 7NS-C12 and 7NS-C38) and pKS-VOTL (clones 7NS-T30, 7NS-T20, 7NS-T8 and 7NS-T4) plasmids, stained with Coomassie brilliant blue R-250 (Bio-Rad). (D) Immunostaining of untransformed 86079/7NS and transformed 86079/7NS with pMAS-LoriC (clone 7NS-C12 and 7NS-C38) and pKS-VOTL (clones 7NS-T30, 7NS-T20, 7NS-8 and 7NS-T4) using polyclonal rabbit serum against M . synoviae Obg_1 peptide. Location of overexpressed Obg is indicated with an arrow on the right. (E) For comparison of Obg expression in MS-H and 86079/7NS, approximately equal amounts (88 μg/lane) of whole cell proteins from MS-H, MS-H-C28, MS-H-T75, 86079/7NS, 7NS-C38 and 7NS-T30 were separated by SDS-PAGE (8.75%) and stained with Coomassie Brilliant Blue. (F) Immunostaining at two different concentrations of whole cell proteins (i.e. 1/3 and 2/3 of that loaded onto SDS-PAGE as shown in panel E) of each strain/transformant using polyclonal rabbit serum against M . synoviae Obg_1 peptide. MW, protein marker (Precision Plus Protein, Dual Color, Bio-Rad).
    Western Blotting, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 162 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc western immunoblotting
    NE, PR3, and MPO localization during NET formation. (A–C) Naive and PMA-activated neutrophils in the presence or absence of NEi, fixed at the indicated time points and immunolabeled for NE (red) and PR3 (green; A), or NE (red) and MPO (green; B and C). DNA was stained with DRAQ5 (blue). (A) NE, and to a lesser extent PR3, translocate to the nucleus within 60 min after stimulation. (B) MPO associates with DNA before cell lysis but later than NE. (C) NEi prevents NE and MPO translocation to the nucleus, and chromatin decondensation. Bar, 5 µm. (D) NE is released from the granules during activation. Lysates from naive and activated neutrophils were prepared after 60 min of incubation and separated into cytoplasmic (HSS) and granule (HSP) fractions. The enzymatic activity (initial rate of change in absorbance) was normalized to the total amount of MPO (MPOt), which remains unchanged, and plotted as the fraction of NE activity over total MPO activity (open bars). The distribution of MPO activity in each sample over total MPO activity is also shown (shaded bars). Samples were also resolved by SDS-PAGE and analyzed by <t>immunoblotting</t> against MPO, NE, and histone H2B.
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    Genzyme western immunoblotting
    NE, PR3, and MPO localization during NET formation. (A–C) Naive and PMA-activated neutrophils in the presence or absence of NEi, fixed at the indicated time points and immunolabeled for NE (red) and PR3 (green; A), or NE (red) and MPO (green; B and C). DNA was stained with DRAQ5 (blue). (A) NE, and to a lesser extent PR3, translocate to the nucleus within 60 min after stimulation. (B) MPO associates with DNA before cell lysis but later than NE. (C) NEi prevents NE and MPO translocation to the nucleus, and chromatin decondensation. Bar, 5 µm. (D) NE is released from the granules during activation. Lysates from naive and activated neutrophils were prepared after 60 min of incubation and separated into cytoplasmic (HSS) and granule (HSP) fractions. The enzymatic activity (initial rate of change in absorbance) was normalized to the total amount of MPO (MPOt), which remains unchanged, and plotted as the fraction of NE activity over total MPO activity (open bars). The distribution of MPO activity in each sample over total MPO activity is also shown (shaded bars). Samples were also resolved by SDS-PAGE and analyzed by <t>immunoblotting</t> against MPO, NE, and histone H2B.
    Western Immunoblotting, supplied by Genzyme, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Immunetics western immunoblot
    NE, PR3, and MPO localization during NET formation. (A–C) Naive and PMA-activated neutrophils in the presence or absence of NEi, fixed at the indicated time points and immunolabeled for NE (red) and PR3 (green; A), or NE (red) and MPO (green; B and C). DNA was stained with DRAQ5 (blue). (A) NE, and to a lesser extent PR3, translocate to the nucleus within 60 min after stimulation. (B) MPO associates with DNA before cell lysis but later than NE. (C) NEi prevents NE and MPO translocation to the nucleus, and chromatin decondensation. Bar, 5 µm. (D) NE is released from the granules during activation. Lysates from naive and activated neutrophils were prepared after 60 min of incubation and separated into cytoplasmic (HSS) and granule (HSP) fractions. The enzymatic activity (initial rate of change in absorbance) was normalized to the total amount of MPO (MPOt), which remains unchanged, and plotted as the fraction of NE activity over total MPO activity (open bars). The distribution of MPO activity in each sample over total MPO activity is also shown (shaded bars). Samples were also resolved by SDS-PAGE and analyzed by <t>immunoblotting</t> against MPO, NE, and histone H2B.
    Western Immunoblot, supplied by Immunetics, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher western immunoblot
    Tfs4 VirD2 interaction with a putative VirC1 homologue. A , gene arrangement in the vicinity of virD2 within the H. pylori P12 tfs4 gene cluster ( 12 ). Genes encoding VirD2 and VirC1 are highlighted by black and dark gray shading , respectively, and the intergenic position of the putative tfs4 oriT sequence is indicated by a triangle. B , pull-down assay demonstrating Tfs4 VirD2-VirC1 protein-protein interaction. Whole-cell lysates containing MBP-VirD2, MBP-VirD2(N), or MBP alone were immobilized on amylose resin, washed thoroughly, and then mixed with a soluble cell lysate containing His-tagged Tfs4 VirC1. Following a further 10× column volume wash step, immobilized prote ins were eluted, and samples were analyzed by 12.5% SDS-PAGE and Western <t>immunoblot</t> using anti-His 6 antibodies (Novagen). His-tagged VirC1 (indicated by an arrow ) is shown to be present in the soluble cell lysate prior to incubation with MBP proteins ( lane 1 ) and is specifically co-eluted with amylose resin-immobilized MBP-VirD2 ( lane 2 ) and MBP-VirD2(N) ( lane 3 ) but not MBP alone ( lane 4 ).
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    GE Healthcare ecf western blotting wb kit
    Tfs4 VirD2 interaction with a putative VirC1 homologue. A , gene arrangement in the vicinity of virD2 within the H. pylori P12 tfs4 gene cluster ( 12 ). Genes encoding VirD2 and VirC1 are highlighted by black and dark gray shading , respectively, and the intergenic position of the putative tfs4 oriT sequence is indicated by a triangle. B , pull-down assay demonstrating Tfs4 VirD2-VirC1 protein-protein interaction. Whole-cell lysates containing MBP-VirD2, MBP-VirD2(N), or MBP alone were immobilized on amylose resin, washed thoroughly, and then mixed with a soluble cell lysate containing His-tagged Tfs4 VirC1. Following a further 10× column volume wash step, immobilized prote ins were eluted, and samples were analyzed by 12.5% SDS-PAGE and Western <t>immunoblot</t> using anti-His 6 antibodies (Novagen). His-tagged VirC1 (indicated by an arrow ) is shown to be present in the soluble cell lysate prior to incubation with MBP proteins ( lane 1 ) and is specifically co-eluted with amylose resin-immobilized MBP-VirD2 ( lane 2 ) and MBP-VirD2(N) ( lane 3 ) but not MBP alone ( lane 4 ).
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    Bio-Rad wb kit
    Tfs4 VirD2 interaction with a putative VirC1 homologue. A , gene arrangement in the vicinity of virD2 within the H. pylori P12 tfs4 gene cluster ( 12 ). Genes encoding VirD2 and VirC1 are highlighted by black and dark gray shading , respectively, and the intergenic position of the putative tfs4 oriT sequence is indicated by a triangle. B , pull-down assay demonstrating Tfs4 VirD2-VirC1 protein-protein interaction. Whole-cell lysates containing MBP-VirD2, MBP-VirD2(N), or MBP alone were immobilized on amylose resin, washed thoroughly, and then mixed with a soluble cell lysate containing His-tagged Tfs4 VirC1. Following a further 10× column volume wash step, immobilized prote ins were eluted, and samples were analyzed by 12.5% SDS-PAGE and Western <t>immunoblot</t> using anti-His 6 antibodies (Novagen). His-tagged VirC1 (indicated by an arrow ) is shown to be present in the soluble cell lysate prior to incubation with MBP proteins ( lane 1 ) and is specifically co-eluted with amylose resin-immobilized MBP-VirD2 ( lane 2 ) and MBP-VirD2(N) ( lane 3 ) but not MBP alone ( lane 4 ).
    Wb Kit, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare immunoblot western analysis
    Supernatant proteins from wild-type and Δ invF strains. Lane 1, supernatant proteins from the secretion defective spaS mutant SVM514; lanes 2 to 7, S. typhimurium SL1344 containing pWSK130, pHD9 ( invF + ), and p hilA (lanes 2 to 4) and the invF mutant SVM579 containing the same plasmids in the same order (lanes 5 to 7); lanes 8 and 9, supernatant proteins from SVM579 strains containing the vector pVLT33 (lane 8) and the IPTG-inducible sigDE clone pHH37 (lane 9). Positions of molecular weight standards are indicated in kilodaltons on the left; previously identified secreted proteins are indicated on the right. Question marks denote proteins that have not been confirmed by <t>immunoblot</t> analysis. Proteins were prepared and analyzed as described in Materials and Methods.
    Immunoblot Western Analysis, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 80/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher western breeze chemiluminescence wb immunodetection kit
    Supernatant proteins from wild-type and Δ invF strains. Lane 1, supernatant proteins from the secretion defective spaS mutant SVM514; lanes 2 to 7, S. typhimurium SL1344 containing pWSK130, pHD9 ( invF + ), and p hilA (lanes 2 to 4) and the invF mutant SVM579 containing the same plasmids in the same order (lanes 5 to 7); lanes 8 and 9, supernatant proteins from SVM579 strains containing the vector pVLT33 (lane 8) and the IPTG-inducible sigDE clone pHH37 (lane 9). Positions of molecular weight standards are indicated in kilodaltons on the left; previously identified secreted proteins are indicated on the right. Question marks denote proteins that have not been confirmed by <t>immunoblot</t> analysis. Proteins were prepared and analyzed as described in Materials and Methods.
    Western Breeze Chemiluminescence Wb Immunodetection Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beyotime western blotting wb
    Supernatant proteins from wild-type and Δ invF strains. Lane 1, supernatant proteins from the secretion defective spaS mutant SVM514; lanes 2 to 7, S. typhimurium SL1344 containing pWSK130, pHD9 ( invF + ), and p hilA (lanes 2 to 4) and the invF mutant SVM579 containing the same plasmids in the same order (lanes 5 to 7); lanes 8 and 9, supernatant proteins from SVM579 strains containing the vector pVLT33 (lane 8) and the IPTG-inducible sigDE clone pHH37 (lane 9). Positions of molecular weight standards are indicated in kilodaltons on the left; previously identified secreted proteins are indicated on the right. Question marks denote proteins that have not been confirmed by <t>immunoblot</t> analysis. Proteins were prepared and analyzed as described in Materials and Methods.
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    Valiant western blot wb
    Supernatant proteins from wild-type and Δ invF strains. Lane 1, supernatant proteins from the secretion defective spaS mutant SVM514; lanes 2 to 7, S. typhimurium SL1344 containing pWSK130, pHD9 ( invF + ), and p hilA (lanes 2 to 4) and the invF mutant SVM579 containing the same plasmids in the same order (lanes 5 to 7); lanes 8 and 9, supernatant proteins from SVM579 strains containing the vector pVLT33 (lane 8) and the IPTG-inducible sigDE clone pHH37 (lane 9). Positions of molecular weight standards are indicated in kilodaltons on the left; previously identified secreted proteins are indicated on the right. Question marks denote proteins that have not been confirmed by <t>immunoblot</t> analysis. Proteins were prepared and analyzed as described in Materials and Methods.
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    Novocastra western immunoblot western immunoblots
    Supernatant proteins from wild-type and Δ invF strains. Lane 1, supernatant proteins from the secretion defective spaS mutant SVM514; lanes 2 to 7, S. typhimurium SL1344 containing pWSK130, pHD9 ( invF + ), and p hilA (lanes 2 to 4) and the invF mutant SVM579 containing the same plasmids in the same order (lanes 5 to 7); lanes 8 and 9, supernatant proteins from SVM579 strains containing the vector pVLT33 (lane 8) and the IPTG-inducible sigDE clone pHH37 (lane 9). Positions of molecular weight standards are indicated in kilodaltons on the left; previously identified secreted proteins are indicated on the right. Question marks denote proteins that have not been confirmed by <t>immunoblot</t> analysis. Proteins were prepared and analyzed as described in Materials and Methods.
    Western Immunoblot Western Immunoblots, supplied by Novocastra, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Trinity Biotech igg western blot wb assays
    Supernatant proteins from wild-type and Δ invF strains. Lane 1, supernatant proteins from the secretion defective spaS mutant SVM514; lanes 2 to 7, S. typhimurium SL1344 containing pWSK130, pHD9 ( invF + ), and p hilA (lanes 2 to 4) and the invF mutant SVM579 containing the same plasmids in the same order (lanes 5 to 7); lanes 8 and 9, supernatant proteins from SVM579 strains containing the vector pVLT33 (lane 8) and the IPTG-inducible sigDE clone pHH37 (lane 9). Positions of molecular weight standards are indicated in kilodaltons on the left; previously identified secreted proteins are indicated on the right. Question marks denote proteins that have not been confirmed by <t>immunoblot</t> analysis. Proteins were prepared and analyzed as described in Materials and Methods.
    Igg Western Blot Wb Assays, supplied by Trinity Biotech, used in various techniques. Bioz Stars score: 93/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Comparison of secretion capacity in the HAP-less and cap-less strains. E. coli MC1000 Δ fliC Δ flgKL (ΔCKL) and Δ fliC D containing the plasmid pTrc-FliC-ΔD3 were grown in LB supplemented with 0.05 mM IPTG, and harvested at OD 600 1.5. Samples which represented either 25 µL or 400 µL culture media were loaded for SDS-PAGE, for intracellular and secreted protein respectively. a Representative images showing the secreted fractions following Coomassie staining, and the intracellular fractions following immunoblotting using an anti-flagellin (H48) antibody and a HRP secondary. A FliC-ΔD3 protein standard (S) was also included, to allow quantification of protein concentration by densitometry ( b , c ). Quantifications for biological triplicates, ± SE and individual data points shown, *p

    Journal: Microbial Cell Factories

    Article Title: Engineering the flagellar type III secretion system: improving capacity for secretion of recombinant protein

    doi: 10.1186/s12934-019-1058-4

    Figure Lengend Snippet: Comparison of secretion capacity in the HAP-less and cap-less strains. E. coli MC1000 Δ fliC Δ flgKL (ΔCKL) and Δ fliC D containing the plasmid pTrc-FliC-ΔD3 were grown in LB supplemented with 0.05 mM IPTG, and harvested at OD 600 1.5. Samples which represented either 25 µL or 400 µL culture media were loaded for SDS-PAGE, for intracellular and secreted protein respectively. a Representative images showing the secreted fractions following Coomassie staining, and the intracellular fractions following immunoblotting using an anti-flagellin (H48) antibody and a HRP secondary. A FliC-ΔD3 protein standard (S) was also included, to allow quantification of protein concentration by densitometry ( b , c ). Quantifications for biological triplicates, ± SE and individual data points shown, *p

    Article Snippet: Gels were either stained with InstantBlue™ (Expedeon) or prepared for Western immunoblotting by electro-transfer onto nitrocellulose membrane (GE Healthcare Life Science) and blocked in 3% w/v bovine serum albumin in TBS (24 g L−1 Tris base, 88 g L−1 NaCl plus 0.1% v/v Tween).

    Techniques: Plasmid Preparation, SDS Page, Staining, Protein Concentration

    Analysis of Obg expression in M . synoviae strains using SDS-PAGE and immunoblotting. (A) SDS-PAGE (7.5%) of whole cell proteins from M . synoviae MS-H transformed with pMAS-LoriC (clone MS-H-C28) and pKS-VOTL (clones MS-H-T75, MS-H-T78 and MS-H-T90) plasmids, stained with Coomassie brilliant blue R-250 (Bio-Rad). Location of ~ 50 kDa protein in pKS-VOTL transformants is indicated with an arrow. (B) Immunostaining of untransformed 86079/7NS, MS-H, and transformed MS-H with pMAS-LoriC (clone MS-H-C28) and pKS-VOTL (clones MS-H-T75, MS-H-T78 and MS-H-T90) using polyclonal rabbit serum against M . synoviae Obg_3 peptide. Location of Obg-MBP fusion protein and overexpressed Obg is indicated with arrows on the right. (C) SDS-PAGE (8.75%) of whole cell proteins from M . synoviae 86079/7NS transformed with pMAS-LoriC (clone 7NS-C12 and 7NS-C38) and pKS-VOTL (clones 7NS-T30, 7NS-T20, 7NS-T8 and 7NS-T4) plasmids, stained with Coomassie brilliant blue R-250 (Bio-Rad). (D) Immunostaining of untransformed 86079/7NS and transformed 86079/7NS with pMAS-LoriC (clone 7NS-C12 and 7NS-C38) and pKS-VOTL (clones 7NS-T30, 7NS-T20, 7NS-8 and 7NS-T4) using polyclonal rabbit serum against M . synoviae Obg_1 peptide. Location of overexpressed Obg is indicated with an arrow on the right. (E) For comparison of Obg expression in MS-H and 86079/7NS, approximately equal amounts (88 μg/lane) of whole cell proteins from MS-H, MS-H-C28, MS-H-T75, 86079/7NS, 7NS-C38 and 7NS-T30 were separated by SDS-PAGE (8.75%) and stained with Coomassie Brilliant Blue. (F) Immunostaining at two different concentrations of whole cell proteins (i.e. 1/3 and 2/3 of that loaded onto SDS-PAGE as shown in panel E) of each strain/transformant using polyclonal rabbit serum against M . synoviae Obg_1 peptide. MW, protein marker (Precision Plus Protein, Dual Color, Bio-Rad).

    Journal: PLoS ONE

    Article Title: Complementation of the Mycoplasma synoviae MS-H vaccine strain with wild-type obg influencing its growth characteristics

    doi: 10.1371/journal.pone.0194528

    Figure Lengend Snippet: Analysis of Obg expression in M . synoviae strains using SDS-PAGE and immunoblotting. (A) SDS-PAGE (7.5%) of whole cell proteins from M . synoviae MS-H transformed with pMAS-LoriC (clone MS-H-C28) and pKS-VOTL (clones MS-H-T75, MS-H-T78 and MS-H-T90) plasmids, stained with Coomassie brilliant blue R-250 (Bio-Rad). Location of ~ 50 kDa protein in pKS-VOTL transformants is indicated with an arrow. (B) Immunostaining of untransformed 86079/7NS, MS-H, and transformed MS-H with pMAS-LoriC (clone MS-H-C28) and pKS-VOTL (clones MS-H-T75, MS-H-T78 and MS-H-T90) using polyclonal rabbit serum against M . synoviae Obg_3 peptide. Location of Obg-MBP fusion protein and overexpressed Obg is indicated with arrows on the right. (C) SDS-PAGE (8.75%) of whole cell proteins from M . synoviae 86079/7NS transformed with pMAS-LoriC (clone 7NS-C12 and 7NS-C38) and pKS-VOTL (clones 7NS-T30, 7NS-T20, 7NS-T8 and 7NS-T4) plasmids, stained with Coomassie brilliant blue R-250 (Bio-Rad). (D) Immunostaining of untransformed 86079/7NS and transformed 86079/7NS with pMAS-LoriC (clone 7NS-C12 and 7NS-C38) and pKS-VOTL (clones 7NS-T30, 7NS-T20, 7NS-8 and 7NS-T4) using polyclonal rabbit serum against M . synoviae Obg_1 peptide. Location of overexpressed Obg is indicated with an arrow on the right. (E) For comparison of Obg expression in MS-H and 86079/7NS, approximately equal amounts (88 μg/lane) of whole cell proteins from MS-H, MS-H-C28, MS-H-T75, 86079/7NS, 7NS-C38 and 7NS-T30 were separated by SDS-PAGE (8.75%) and stained with Coomassie Brilliant Blue. (F) Immunostaining at two different concentrations of whole cell proteins (i.e. 1/3 and 2/3 of that loaded onto SDS-PAGE as shown in panel E) of each strain/transformant using polyclonal rabbit serum against M . synoviae Obg_1 peptide. MW, protein marker (Precision Plus Protein, Dual Color, Bio-Rad).

    Article Snippet: For Western immunoblotting, unstained gels were used for protein transfer onto PVDF membrane using Trans-Blot Turbo RTA Mini PVDF Transfer Kit (Bio-Rad) using Trans-Blot Turbo Transfer System (Bio-Rad).

    Techniques: Expressing, SDS Page, Mass Spectrometry, Transformation Assay, Staining, Immunostaining, Marker

    The effects of Nrf2 modulation on tumour growth in vivo (A) Example IVIS images of BALB/c mice from the brusatol treated and control groups after subcutaneous flank injection of the CT26lucA6c cell line. (B) Graphical display of the significant inhibition of tumour growth in mice treated with brusatol in comparison to vehicle treated controls (multiple t -tests, N=3/group, mean +/-SEM). (C) Photos of the flank tumours excised from mice (scale in 1mm increments) 21 days after implantation. (D) Western immunoblotting and densitometry confirmed significant inhibition of Nrf2 in the flank tumours excised from mice treated with brusatol (p=0.03, unpaired t -test, bar chart displays mean and standard deviation). (E) Example serial IVIS ® images of BALB/c mice orthotopically injected with the CT26lucA6c cell line with time from implantation of cells. (F) Data displayed graphically as fold change in luminescence from the first day of treatment. All treatments inhibited tumour growth significantly compared to mice in the control group (one-way ANOVA, N=8, graphs display mean +/- SEM). (G) Representative images from tissue stained for Nrf2 by IHC. Moderate to strong diffuse staining is demonstrated in the a) positive control of liver from BALB/c mice treated with CDDO-me. b) Livers from Nrf2 knockout mice were used as a negative control and were absent of Nrf2 staining. There was strong staining demonstrated in caecal tumours excised from mice in the c) control group and staining was reduced in the tumours from mice treated with d) brusatol. Graphical display of Nrf2 staining semi-quantification reveals significantly (p=0.04 unpaired t -test with Welch’s correction) reduced Nrf2 staining semi-quantification in brusatol treated mice (N=3, graph displays mean +/- SD). C = control, BRU = brusatol, IR = irinotecan.

    Journal: Oncotarget

    Article Title: The Nrf2 inhibitor brusatol is a potent antitumour agent in an orthotopic mouse model of colorectal cancer

    doi: 10.18632/oncotarget.25497

    Figure Lengend Snippet: The effects of Nrf2 modulation on tumour growth in vivo (A) Example IVIS images of BALB/c mice from the brusatol treated and control groups after subcutaneous flank injection of the CT26lucA6c cell line. (B) Graphical display of the significant inhibition of tumour growth in mice treated with brusatol in comparison to vehicle treated controls (multiple t -tests, N=3/group, mean +/-SEM). (C) Photos of the flank tumours excised from mice (scale in 1mm increments) 21 days after implantation. (D) Western immunoblotting and densitometry confirmed significant inhibition of Nrf2 in the flank tumours excised from mice treated with brusatol (p=0.03, unpaired t -test, bar chart displays mean and standard deviation). (E) Example serial IVIS ® images of BALB/c mice orthotopically injected with the CT26lucA6c cell line with time from implantation of cells. (F) Data displayed graphically as fold change in luminescence from the first day of treatment. All treatments inhibited tumour growth significantly compared to mice in the control group (one-way ANOVA, N=8, graphs display mean +/- SEM). (G) Representative images from tissue stained for Nrf2 by IHC. Moderate to strong diffuse staining is demonstrated in the a) positive control of liver from BALB/c mice treated with CDDO-me. b) Livers from Nrf2 knockout mice were used as a negative control and were absent of Nrf2 staining. There was strong staining demonstrated in caecal tumours excised from mice in the c) control group and staining was reduced in the tumours from mice treated with d) brusatol. Graphical display of Nrf2 staining semi-quantification reveals significantly (p=0.04 unpaired t -test with Welch’s correction) reduced Nrf2 staining semi-quantification in brusatol treated mice (N=3, graph displays mean +/- SD). C = control, BRU = brusatol, IR = irinotecan.

    Article Snippet: Western immunoblotting Western immunoblotting was undertaken on 40 micrograms of cell lysate and tumour tissue homogenate were loaded in Laemmli buffer (BioRad, Hercules, CA) on Mini-PRTOEAN® TGX™ Precast Gels (BioRad, Hercules, CA).

    Techniques: In Vivo, Mouse Assay, Injection, Inhibition, Western Blot, Standard Deviation, Staining, Immunohistochemistry, Positive Control, Knock-Out, Negative Control

    NE, PR3, and MPO localization during NET formation. (A–C) Naive and PMA-activated neutrophils in the presence or absence of NEi, fixed at the indicated time points and immunolabeled for NE (red) and PR3 (green; A), or NE (red) and MPO (green; B and C). DNA was stained with DRAQ5 (blue). (A) NE, and to a lesser extent PR3, translocate to the nucleus within 60 min after stimulation. (B) MPO associates with DNA before cell lysis but later than NE. (C) NEi prevents NE and MPO translocation to the nucleus, and chromatin decondensation. Bar, 5 µm. (D) NE is released from the granules during activation. Lysates from naive and activated neutrophils were prepared after 60 min of incubation and separated into cytoplasmic (HSS) and granule (HSP) fractions. The enzymatic activity (initial rate of change in absorbance) was normalized to the total amount of MPO (MPOt), which remains unchanged, and plotted as the fraction of NE activity over total MPO activity (open bars). The distribution of MPO activity in each sample over total MPO activity is also shown (shaded bars). Samples were also resolved by SDS-PAGE and analyzed by immunoblotting against MPO, NE, and histone H2B.

    Journal: The Journal of Cell Biology

    Article Title: Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps

    doi: 10.1083/jcb.201006052

    Figure Lengend Snippet: NE, PR3, and MPO localization during NET formation. (A–C) Naive and PMA-activated neutrophils in the presence or absence of NEi, fixed at the indicated time points and immunolabeled for NE (red) and PR3 (green; A), or NE (red) and MPO (green; B and C). DNA was stained with DRAQ5 (blue). (A) NE, and to a lesser extent PR3, translocate to the nucleus within 60 min after stimulation. (B) MPO associates with DNA before cell lysis but later than NE. (C) NEi prevents NE and MPO translocation to the nucleus, and chromatin decondensation. Bar, 5 µm. (D) NE is released from the granules during activation. Lysates from naive and activated neutrophils were prepared after 60 min of incubation and separated into cytoplasmic (HSS) and granule (HSP) fractions. The enzymatic activity (initial rate of change in absorbance) was normalized to the total amount of MPO (MPOt), which remains unchanged, and plotted as the fraction of NE activity over total MPO activity (open bars). The distribution of MPO activity in each sample over total MPO activity is also shown (shaded bars). Samples were also resolved by SDS-PAGE and analyzed by immunoblotting against MPO, NE, and histone H2B.

    Article Snippet: Antibodies for Western immunoblotting 1:1,000 rabbit anti-H2A (2578, recognizes the C terminus; Cell Signaling Technology), 1:5,000 anti-H2B (07-371, recognizes aa 118–126; Millipore), 1:10,000 anti-H3 (07-690, recognizes the C terminus; Millipore), 1:5,000 anti-H4 (04-858, epitope mapped to aa 25–28; Millipore), and the pan-histone antibody MAB052 (1:500; Millipore) for analysis of H1.

    Techniques: Immunolabeling, Staining, Lysis, Translocation Assay, Activation Assay, Incubation, Activity Assay, SDS Page

    NE partially degrades core histones during NET formation. (A) Histone cleavage during NET formation is inhibited by NEi. Western immunoblotting against histones in lysates of naive (N) and PMA-activated neutrophils (PMA) in the presence (+NEi) or in the absence of NEi (untreated). (B and C) Quantitation of chromatin decondensation for the samples shown in A. (B) Untreated neutrophils. (C) Neutrophils treated with NEi. Shown are naive neutrophils at 0 h (gray) or 4 h (black), activated with PMA for 1 (yellow), 2 (orange), 3 (red), or 4 h (blue). (D) NE is not significantly externalized before NET formation. The time course of the release of NE into the medium by neutrophils activated with PMA measured by ELISA is shown. MNase was added to solubilize NE bound to DNA. Samples were normalized to NE levels from plated naive neutrophils lysed with 0.1% Triton X-100 (Total).

    Journal: The Journal of Cell Biology

    Article Title: Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps

    doi: 10.1083/jcb.201006052

    Figure Lengend Snippet: NE partially degrades core histones during NET formation. (A) Histone cleavage during NET formation is inhibited by NEi. Western immunoblotting against histones in lysates of naive (N) and PMA-activated neutrophils (PMA) in the presence (+NEi) or in the absence of NEi (untreated). (B and C) Quantitation of chromatin decondensation for the samples shown in A. (B) Untreated neutrophils. (C) Neutrophils treated with NEi. Shown are naive neutrophils at 0 h (gray) or 4 h (black), activated with PMA for 1 (yellow), 2 (orange), 3 (red), or 4 h (blue). (D) NE is not significantly externalized before NET formation. The time course of the release of NE into the medium by neutrophils activated with PMA measured by ELISA is shown. MNase was added to solubilize NE bound to DNA. Samples were normalized to NE levels from plated naive neutrophils lysed with 0.1% Triton X-100 (Total).

    Article Snippet: Antibodies for Western immunoblotting 1:1,000 rabbit anti-H2A (2578, recognizes the C terminus; Cell Signaling Technology), 1:5,000 anti-H2B (07-371, recognizes aa 118–126; Millipore), 1:10,000 anti-H3 (07-690, recognizes the C terminus; Millipore), 1:5,000 anti-H4 (04-858, epitope mapped to aa 25–28; Millipore), and the pan-histone antibody MAB052 (1:500; Millipore) for analysis of H1.

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

    NE cleaves histones and promotes nuclear decondensation in vitro. (A) Nuclei isolated from neutrophils were incubated in buffer or in neutrophil-derived LSS lysates for 120 min at 37°C and labeled with Sytox green. Bar, 10 µm. (B) Neutrophil extracts are sufficient to decondense nuclei from other cell types. Nuclear decondensation of LSS extracts from HL-60 cells differentiated with RA, HeLa cells, PBMCs, and neutrophils were tested with nuclei isolated from neutrophils (Neut). The nuclear area was quantified using ImageJ image processing software. Circles denote the median area and the bars indicate the range of the nuclear area values, calculated from the standard deviation of each dataset. (C) Decondensation activity is stored in azurophilic granules. Decondensation of nuclei treated with buffer, neutrophil LSS, neutrophil HSS, HSP, and the granular subfractions of gelatinase (1), specific (2), and azurophilic (3) granules were purified over a discontinuous Percoll gradient. Nuclei were also incubated with LSS or HSP in the presence or absence of NEi, SLPI, CGi, or ABAH. Inhibitors were used at the indicated concentrations. (D) The purity of gelatinase (1), specific (2), and azurophilic (3) granule fractions was tested by SDS-PAGE immunoblot analysis using antibodies against gelatinase, lactoferrin, MPO, and NE. NE fractionates exclusively with azurophilic granules (3). (E) NEi inhibits the degradation of nuclear H4 in vitro. After 120 min at 37°C, decondensation reactions were passed over a 0.65-µm filter to separate the nuclei from the extract. The total reaction (T), flowthrough (SF), or nuclear (NF) fractions were analyzed by immunoblotting. Top, nuclei in buffer; middle, nuclei in LSS; bottom, nuclei incubated with LSS and 5 µM NEi. (F) NE and MPO translocate to nuclei in vitro. LSS (L), nuclei alone (N), or LSS + nuclei (L+N), were incubated for the indicated time and separated over filters as in E. The levels of H1, H4, NE, and MPO in the nuclear fraction at different time points were analyzed by immunoblotting. (G) A plot of the corresponding nuclear decondensation, measured as in A, for the experiment in F. (H) Purified NE is sufficient to decondense nuclei in vitro. Nuclei were incubated with buffer (B) or 1 µM purified NE (NE) in the absence of MPO (no MPO, open circles) for the indicated duration (0–240 min). In parallel, 1 µM MPO was added to the same samples (+MPO, closed circles). Decondensation was measured by quantifying the nuclear area and was significantly enhanced in the presence of both NE and MPO (closed circles). (I) Purified NE cleaves nuclear histones in vitro. The samples from H along with NE incubated with purified neutrophil histones were resolved by SDS-PAGE and analyzed by immunoblotting. H4 was processively degraded but H2A, H2B, and H3 were only partially degraded by NE. Soluble purified histones were completely degraded. Notably, the addition of 1 µM MPO had no effect on histone degradation by NE (third column). ***, P

    Journal: The Journal of Cell Biology

    Article Title: Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps

    doi: 10.1083/jcb.201006052

    Figure Lengend Snippet: NE cleaves histones and promotes nuclear decondensation in vitro. (A) Nuclei isolated from neutrophils were incubated in buffer or in neutrophil-derived LSS lysates for 120 min at 37°C and labeled with Sytox green. Bar, 10 µm. (B) Neutrophil extracts are sufficient to decondense nuclei from other cell types. Nuclear decondensation of LSS extracts from HL-60 cells differentiated with RA, HeLa cells, PBMCs, and neutrophils were tested with nuclei isolated from neutrophils (Neut). The nuclear area was quantified using ImageJ image processing software. Circles denote the median area and the bars indicate the range of the nuclear area values, calculated from the standard deviation of each dataset. (C) Decondensation activity is stored in azurophilic granules. Decondensation of nuclei treated with buffer, neutrophil LSS, neutrophil HSS, HSP, and the granular subfractions of gelatinase (1), specific (2), and azurophilic (3) granules were purified over a discontinuous Percoll gradient. Nuclei were also incubated with LSS or HSP in the presence or absence of NEi, SLPI, CGi, or ABAH. Inhibitors were used at the indicated concentrations. (D) The purity of gelatinase (1), specific (2), and azurophilic (3) granule fractions was tested by SDS-PAGE immunoblot analysis using antibodies against gelatinase, lactoferrin, MPO, and NE. NE fractionates exclusively with azurophilic granules (3). (E) NEi inhibits the degradation of nuclear H4 in vitro. After 120 min at 37°C, decondensation reactions were passed over a 0.65-µm filter to separate the nuclei from the extract. The total reaction (T), flowthrough (SF), or nuclear (NF) fractions were analyzed by immunoblotting. Top, nuclei in buffer; middle, nuclei in LSS; bottom, nuclei incubated with LSS and 5 µM NEi. (F) NE and MPO translocate to nuclei in vitro. LSS (L), nuclei alone (N), or LSS + nuclei (L+N), were incubated for the indicated time and separated over filters as in E. The levels of H1, H4, NE, and MPO in the nuclear fraction at different time points were analyzed by immunoblotting. (G) A plot of the corresponding nuclear decondensation, measured as in A, for the experiment in F. (H) Purified NE is sufficient to decondense nuclei in vitro. Nuclei were incubated with buffer (B) or 1 µM purified NE (NE) in the absence of MPO (no MPO, open circles) for the indicated duration (0–240 min). In parallel, 1 µM MPO was added to the same samples (+MPO, closed circles). Decondensation was measured by quantifying the nuclear area and was significantly enhanced in the presence of both NE and MPO (closed circles). (I) Purified NE cleaves nuclear histones in vitro. The samples from H along with NE incubated with purified neutrophil histones were resolved by SDS-PAGE and analyzed by immunoblotting. H4 was processively degraded but H2A, H2B, and H3 were only partially degraded by NE. Soluble purified histones were completely degraded. Notably, the addition of 1 µM MPO had no effect on histone degradation by NE (third column). ***, P

    Article Snippet: Antibodies for Western immunoblotting 1:1,000 rabbit anti-H2A (2578, recognizes the C terminus; Cell Signaling Technology), 1:5,000 anti-H2B (07-371, recognizes aa 118–126; Millipore), 1:10,000 anti-H3 (07-690, recognizes the C terminus; Millipore), 1:5,000 anti-H4 (04-858, epitope mapped to aa 25–28; Millipore), and the pan-histone antibody MAB052 (1:500; Millipore) for analysis of H1.

    Techniques: In Vitro, Isolation, Incubation, Derivative Assay, Labeling, Software, Standard Deviation, Activity Assay, Purification, SDS Page

    Tfs4 VirD2 interaction with a putative VirC1 homologue. A , gene arrangement in the vicinity of virD2 within the H. pylori P12 tfs4 gene cluster ( 12 ). Genes encoding VirD2 and VirC1 are highlighted by black and dark gray shading , respectively, and the intergenic position of the putative tfs4 oriT sequence is indicated by a triangle. B , pull-down assay demonstrating Tfs4 VirD2-VirC1 protein-protein interaction. Whole-cell lysates containing MBP-VirD2, MBP-VirD2(N), or MBP alone were immobilized on amylose resin, washed thoroughly, and then mixed with a soluble cell lysate containing His-tagged Tfs4 VirC1. Following a further 10× column volume wash step, immobilized prote ins were eluted, and samples were analyzed by 12.5% SDS-PAGE and Western immunoblot using anti-His 6 antibodies (Novagen). His-tagged VirC1 (indicated by an arrow ) is shown to be present in the soluble cell lysate prior to incubation with MBP proteins ( lane 1 ) and is specifically co-eluted with amylose resin-immobilized MBP-VirD2 ( lane 2 ) and MBP-VirD2(N) ( lane 3 ) but not MBP alone ( lane 4 ).

    Journal: The Journal of Biological Chemistry

    Article Title: Site-specific Relaxase Activity of a VirD2-like Protein Encoded within the tfs4 Genomic Island of Helicobacter pylori *

    doi: 10.1074/jbc.M113.496430

    Figure Lengend Snippet: Tfs4 VirD2 interaction with a putative VirC1 homologue. A , gene arrangement in the vicinity of virD2 within the H. pylori P12 tfs4 gene cluster ( 12 ). Genes encoding VirD2 and VirC1 are highlighted by black and dark gray shading , respectively, and the intergenic position of the putative tfs4 oriT sequence is indicated by a triangle. B , pull-down assay demonstrating Tfs4 VirD2-VirC1 protein-protein interaction. Whole-cell lysates containing MBP-VirD2, MBP-VirD2(N), or MBP alone were immobilized on amylose resin, washed thoroughly, and then mixed with a soluble cell lysate containing His-tagged Tfs4 VirC1. Following a further 10× column volume wash step, immobilized prote ins were eluted, and samples were analyzed by 12.5% SDS-PAGE and Western immunoblot using anti-His 6 antibodies (Novagen). His-tagged VirC1 (indicated by an arrow ) is shown to be present in the soluble cell lysate prior to incubation with MBP proteins ( lane 1 ) and is specifically co-eluted with amylose resin-immobilized MBP-VirD2 ( lane 2 ) and MBP-VirD2(N) ( lane 3 ) but not MBP alone ( lane 4 ).

    Article Snippet: Immobilized proteins were finally eluted by the addition of 35 μl of buffer A containing maltose, and then 10 μl was resolved by 12.5% SDS-PAGE (Invitrogen) prior to Western immunoblot.

    Techniques: Sequencing, Pull Down Assay, SDS Page, Western Blot, Incubation

    Supernatant proteins from wild-type and Δ invF strains. Lane 1, supernatant proteins from the secretion defective spaS mutant SVM514; lanes 2 to 7, S. typhimurium SL1344 containing pWSK130, pHD9 ( invF + ), and p hilA (lanes 2 to 4) and the invF mutant SVM579 containing the same plasmids in the same order (lanes 5 to 7); lanes 8 and 9, supernatant proteins from SVM579 strains containing the vector pVLT33 (lane 8) and the IPTG-inducible sigDE clone pHH37 (lane 9). Positions of molecular weight standards are indicated in kilodaltons on the left; previously identified secreted proteins are indicated on the right. Question marks denote proteins that have not been confirmed by immunoblot analysis. Proteins were prepared and analyzed as described in Materials and Methods.

    Journal: Journal of Bacteriology

    Article Title: InvF Is Required for Expression of Genes Encoding Proteins Secreted by the SPI1 Type III Secretion Apparatus in Salmonella typhimurium

    doi:

    Figure Lengend Snippet: Supernatant proteins from wild-type and Δ invF strains. Lane 1, supernatant proteins from the secretion defective spaS mutant SVM514; lanes 2 to 7, S. typhimurium SL1344 containing pWSK130, pHD9 ( invF + ), and p hilA (lanes 2 to 4) and the invF mutant SVM579 containing the same plasmids in the same order (lanes 5 to 7); lanes 8 and 9, supernatant proteins from SVM579 strains containing the vector pVLT33 (lane 8) and the IPTG-inducible sigDE clone pHH37 (lane 9). Positions of molecular weight standards are indicated in kilodaltons on the left; previously identified secreted proteins are indicated on the right. Question marks denote proteins that have not been confirmed by immunoblot analysis. Proteins were prepared and analyzed as described in Materials and Methods.

    Article Snippet: Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) ( ) on 7.5% gels for silver stain analysis ( ) and 5% gels for immunoblot (Western) analysis (ECL Western blotting detection system; Amersham Pharmacia Biotech).

    Techniques: Mutagenesis, Plasmid Preparation, Molecular Weight