hrp-linked antibody Search Results


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  • 99
    Thermo Fisher horseradish peroxidase linked secondary antibody
    BADH enzyme activity and BADH expression in control and pDD -Dc-aad A /badh lines. A, A pDD -Dc-aad A /badh transgenic line shown with taproot and shoot. B, BADH activity in untransformed (U) and transformed (T) cell suspension, root, and leaf. Note: Mean and errors bars are the average of three replicates. C, Western blot using polyclonal anti-BADH serum. Antigenic peptides were detected using <t>horseradish</t> <t>peroxidase-linked</t> <t>secondary</t> <t>antibody.</t> Lanes 1 to 3, Untransformed cell culture, root, and leaf; lanes 4 to 6, transformed cell culture, root, and leaf.
    Horseradish Peroxidase Linked Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 67 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/horseradish peroxidase linked secondary antibody/product/Thermo Fisher
    Average 99 stars, based on 67 article reviews
    Price from $9.99 to $1999.99
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    94
    Cell Signaling Technology Inc hrp linked antibody
    HDAC11 knockdown affects acetylation levels of histone H3 in T. castaneum . (A) The total protein extracted from dsHDAC11 or dsmalE injected larvae were resolved on SDS-PAGE gels, transferred to western blots, and probed with antibodies recognizing Acetyl-Histone H3 (Antibody Sampler Kit # 9927-Cell Signaling (H3K9, H3K14, H3K18, H3K27, and H3K56). ß-actin served as a loading control. The <t>HRP-linked</t> <t>IgG</t> (#7074-Cell Signaling) was used as a secondary antibody. Band densities were determined by Image-J software and normalized with loading control protein-ß-Actin. The Mean + SE ( n = 3) band densities are shown. Means marked with different letters are significantly different from each other, P ≤ 0.05 by ANOVA. (B) Acetylated-Lysine (Ac-K 2 -100) MultiMab TM Rabbit mAb mix #9814 was used to detect acetylation levels of proteins extracted from TcA cells exposed to dsHDAC11 or dsmalE. The acetylation levels of histone H3K9, H3K18 and H3K27 increased in HDAC11 knockdown cells were detected as described in Figure 7A . The band densities were quantified and plotted as described in Figure 7A . The Mean + SE ( n = 3) band densities are shown. Means marked with different letters are significantly different from each other, P ≤ 0.05 by ANOVA.
    Hrp Linked Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 2885 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hrp linked antibody/product/Cell Signaling Technology Inc
    Average 94 stars, based on 2885 article reviews
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    99
    Cell Signaling Technology Inc anti rabbit igg hrp linked antibodies
    PRX-1 promotes phosphorylation of STAT3. Whole protein was extracted through RIPA buffer and after performing SDS-PAGE electrophoresis, the protein was transferred to a PVDF membrane. The membrane was subsequently blocked and blots were visualized using antibodies for β-actin, phosphor-STAT3 and STAT3 and anti-mouse <t>IgG</t> or anti-rabbit IgG <t>HRP-linked</t> antibodies.
    Anti Rabbit Igg Hrp Linked Antibodies, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1947 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti rabbit igg hrp linked antibodies/product/Cell Signaling Technology Inc
    Average 99 stars, based on 1947 article reviews
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    99
    Cell Signaling Technology Inc anti mouse igg hrp linked antibody
    PRX-1 promotes phosphorylation of STAT3. Whole protein was extracted through RIPA buffer and after performing SDS-PAGE electrophoresis, the protein was transferred to a PVDF membrane. The membrane was subsequently blocked and blots were visualized using antibodies for β-actin, phosphor-STAT3 and STAT3 and anti-mouse <t>IgG</t> or anti-rabbit IgG <t>HRP-linked</t> antibodies.
    Anti Mouse Igg Hrp Linked Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 841 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti mouse igg hrp linked antibody/product/Cell Signaling Technology Inc
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    99
    Cell Signaling Technology Inc anti mouse
    Protein phosphorylation associated with myometrial contractility. Myometrial strips were snap frozen at specific stages during the development of contractions. The tissue was then pulverised, protein extracted and separated by 1D SDS-PAGE. (A) Phosphorylated myometrial proteins were labelled in gel with Pro-Q® Diamond Phospho Stain and visualised using a FujiFilm LAS-3000 luminescent image analyser. The gel was then SYPRO® Ruby Protein stained to visualise total protein and ascertain equal loading. Lanes M P and M T illustrate the Peppermint™ Stick molecular weight marker labelled with Pro-Q® Diamond Stain and SYPRO Ruby Protein Stain respectively. Dotted line indicates removal on none-relevant gel lanes. (B) Separated proteins were western transferred to nitrocellulose membrane. Total protein loading was visualised using SYPRO Ruby Blot Stain and equal loading across lanes confirmed. Membranes were then probed with mouse anti-phosphotyrosine (1∶2500) and anti-mouse <t>IgG-HRP</t> (1∶2500). The immunoreactive bands were detected using a FujiFilm LAS-3000 luminescent image analyser. Positive and negative controls were protein extracts from capacitated and non-capacitated human spermatozoa respectively. Black arrowheads and white arrowheads indicate phosphorylation events associated with contraction and relaxation respectively. (i), (ii) and (iv) highlight bands exhibiting phasic phosphorylation, whilst phosphorylation remained increased for bands (iii) and (v). Representative images shown from 3 replicates. M r = relative molecular mass×1000. M T = molecular weight marker stained for total protein detection, M P = molecular weight marker stained for phospho protein detection.
    Anti Mouse, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 4344 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare hrp linked whole antibody
    Effects of hypoxia on StAR, <t>P450scc</t> and 3β-HSD protein by non-luteinizing and luteinizing granulosa cells. The cells were cultured under 20% O 2 or 10% O 2 (A and B) and 20% O 2 or 5% O 2 (C and D) for 24 h in the presence or absence of insulin (2 µg/ml), forskolin (10 µM) or insulin (2 µg/ml) in combination with forskolin (10 µM). Representative samples of Western blotting for StAR, P450scc, 3β-HSD and β-actin are shown in Fig. 5A for 10% O 2 and in Fig. 5C for 5% O 2 . The blot was incubated with primary antibodies against StAR, P450scc, 3β-HSD or β-actin and then incubated with secondary antibody conjugated to <t>HRP.</t> The resultant signal was detected by chemiluminescence and quantitated by computer-assisted densitometry. All protein levels are expressed relative to the amounts of β-actin. Asterisks indicate significant differences (P
    Hrp Linked Whole Antibody, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 401 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hrp linked whole antibody/product/GE Healthcare
    Average 99 stars, based on 401 article reviews
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    99
    GE Healthcare hrp linked secondary antibodies
    Effects of hypoxia on StAR, <t>P450scc</t> and 3β-HSD protein by non-luteinizing and luteinizing granulosa cells. The cells were cultured under 20% O 2 or 10% O 2 (A and B) and 20% O 2 or 5% O 2 (C and D) for 24 h in the presence or absence of insulin (2 µg/ml), forskolin (10 µM) or insulin (2 µg/ml) in combination with forskolin (10 µM). Representative samples of Western blotting for StAR, P450scc, 3β-HSD and β-actin are shown in Fig. 5A for 10% O 2 and in Fig. 5C for 5% O 2 . The blot was incubated with primary antibodies against StAR, P450scc, 3β-HSD or β-actin and then incubated with secondary antibody conjugated to <t>HRP.</t> The resultant signal was detected by chemiluminescence and quantitated by computer-assisted densitometry. All protein levels are expressed relative to the amounts of β-actin. Asterisks indicate significant differences (P
    Hrp Linked Secondary Antibodies, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 903 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hrp linked secondary antibodies/product/GE Healthcare
    Average 99 stars, based on 903 article reviews
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    92
    Santa Cruz Biotechnology hrp linked secondary antibody
    Effects of hypoxia on StAR, <t>P450scc</t> and 3β-HSD protein by non-luteinizing and luteinizing granulosa cells. The cells were cultured under 20% O 2 or 10% O 2 (A and B) and 20% O 2 or 5% O 2 (C and D) for 24 h in the presence or absence of insulin (2 µg/ml), forskolin (10 µM) or insulin (2 µg/ml) in combination with forskolin (10 µM). Representative samples of Western blotting for StAR, P450scc, 3β-HSD and β-actin are shown in Fig. 5A for 10% O 2 and in Fig. 5C for 5% O 2 . The blot was incubated with primary antibodies against StAR, P450scc, 3β-HSD or β-actin and then incubated with secondary antibody conjugated to <t>HRP.</t> The resultant signal was detected by chemiluminescence and quantitated by computer-assisted densitometry. All protein levels are expressed relative to the amounts of β-actin. Asterisks indicate significant differences (P
    Hrp Linked Secondary Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 98 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hrp linked secondary antibody/product/Santa Cruz Biotechnology
    Average 92 stars, based on 98 article reviews
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    91
    Cell Signaling Technology Inc hrp linked anti rabbit
    Effects of hypoxia on StAR, <t>P450scc</t> and 3β-HSD protein by non-luteinizing and luteinizing granulosa cells. The cells were cultured under 20% O 2 or 10% O 2 (A and B) and 20% O 2 or 5% O 2 (C and D) for 24 h in the presence or absence of insulin (2 µg/ml), forskolin (10 µM) or insulin (2 µg/ml) in combination with forskolin (10 µM). Representative samples of Western blotting for StAR, P450scc, 3β-HSD and β-actin are shown in Fig. 5A for 10% O 2 and in Fig. 5C for 5% O 2 . The blot was incubated with primary antibodies against StAR, P450scc, 3β-HSD or β-actin and then incubated with secondary antibody conjugated to <t>HRP.</t> The resultant signal was detected by chemiluminescence and quantitated by computer-assisted densitometry. All protein levels are expressed relative to the amounts of β-actin. Asterisks indicate significant differences (P
    Hrp Linked Anti Rabbit, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 302 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hrp linked anti rabbit/product/Cell Signaling Technology Inc
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    99
    Bio-Rad horseradish peroxidase linked secondary antibody
    Effects of hypoxia on StAR, <t>P450scc</t> and 3β-HSD protein by non-luteinizing and luteinizing granulosa cells. The cells were cultured under 20% O 2 or 10% O 2 (A and B) and 20% O 2 or 5% O 2 (C and D) for 24 h in the presence or absence of insulin (2 µg/ml), forskolin (10 µM) or insulin (2 µg/ml) in combination with forskolin (10 µM). Representative samples of Western blotting for StAR, P450scc, 3β-HSD and β-actin are shown in Fig. 5A for 10% O 2 and in Fig. 5C for 5% O 2 . The blot was incubated with primary antibodies against StAR, P450scc, 3β-HSD or β-actin and then incubated with secondary antibody conjugated to <t>HRP.</t> The resultant signal was detected by chemiluminescence and quantitated by computer-assisted densitometry. All protein levels are expressed relative to the amounts of β-actin. Asterisks indicate significant differences (P
    Horseradish Peroxidase Linked Secondary Antibody, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/horseradish peroxidase linked secondary antibody/product/Bio-Rad
    Average 99 stars, based on 62 article reviews
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    horseradish peroxidase linked secondary antibody - by Bioz Stars, 2020-09
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    93
    Agilent technologies hrp linked secondary antibodies
    Effects of hypoxia on StAR, <t>P450scc</t> and 3β-HSD protein by non-luteinizing and luteinizing granulosa cells. The cells were cultured under 20% O 2 or 10% O 2 (A and B) and 20% O 2 or 5% O 2 (C and D) for 24 h in the presence or absence of insulin (2 µg/ml), forskolin (10 µM) or insulin (2 µg/ml) in combination with forskolin (10 µM). Representative samples of Western blotting for StAR, P450scc, 3β-HSD and β-actin are shown in Fig. 5A for 10% O 2 and in Fig. 5C for 5% O 2 . The blot was incubated with primary antibodies against StAR, P450scc, 3β-HSD or β-actin and then incubated with secondary antibody conjugated to <t>HRP.</t> The resultant signal was detected by chemiluminescence and quantitated by computer-assisted densitometry. All protein levels are expressed relative to the amounts of β-actin. Asterisks indicate significant differences (P
    Hrp Linked Secondary Antibodies, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 93/100, based on 144 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hrp linked secondary antibodies/product/Agilent technologies
    Average 93 stars, based on 144 article reviews
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    hrp linked secondary antibodies - by Bioz Stars, 2020-09
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    99
    Cell Signaling Technology Inc horseradish peroxidase hrp linked secondary antibodies
    Effects of hypoxia on StAR, <t>P450scc</t> and 3β-HSD protein by non-luteinizing and luteinizing granulosa cells. The cells were cultured under 20% O 2 or 10% O 2 (A and B) and 20% O 2 or 5% O 2 (C and D) for 24 h in the presence or absence of insulin (2 µg/ml), forskolin (10 µM) or insulin (2 µg/ml) in combination with forskolin (10 µM). Representative samples of Western blotting for StAR, P450scc, 3β-HSD and β-actin are shown in Fig. 5A for 10% O 2 and in Fig. 5C for 5% O 2 . The blot was incubated with primary antibodies against StAR, P450scc, 3β-HSD or β-actin and then incubated with secondary antibody conjugated to <t>HRP.</t> The resultant signal was detected by chemiluminescence and quantitated by computer-assisted densitometry. All protein levels are expressed relative to the amounts of β-actin. Asterisks indicate significant differences (P
    Horseradish Peroxidase Hrp Linked Secondary Antibodies, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 177 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/horseradish peroxidase hrp linked secondary antibodies/product/Cell Signaling Technology Inc
    Average 99 stars, based on 177 article reviews
    Price from $9.99 to $1999.99
    horseradish peroxidase hrp linked secondary antibodies - by Bioz Stars, 2020-09
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    Image Search Results


    BADH enzyme activity and BADH expression in control and pDD -Dc-aad A /badh lines. A, A pDD -Dc-aad A /badh transgenic line shown with taproot and shoot. B, BADH activity in untransformed (U) and transformed (T) cell suspension, root, and leaf. Note: Mean and errors bars are the average of three replicates. C, Western blot using polyclonal anti-BADH serum. Antigenic peptides were detected using horseradish peroxidase-linked secondary antibody. Lanes 1 to 3, Untransformed cell culture, root, and leaf; lanes 4 to 6, transformed cell culture, root, and leaf.

    Journal: Plant Physiology

    Article Title: Plastid-Expressed Betaine Aldehyde Dehydrogenase Gene in Carrot Cultured Cells, Roots, and Leaves Confers Enhanced Salt Tolerance 1

    doi: 10.1104/pp.104.045187

    Figure Lengend Snippet: BADH enzyme activity and BADH expression in control and pDD -Dc-aad A /badh lines. A, A pDD -Dc-aad A /badh transgenic line shown with taproot and shoot. B, BADH activity in untransformed (U) and transformed (T) cell suspension, root, and leaf. Note: Mean and errors bars are the average of three replicates. C, Western blot using polyclonal anti-BADH serum. Antigenic peptides were detected using horseradish peroxidase-linked secondary antibody. Lanes 1 to 3, Untransformed cell culture, root, and leaf; lanes 4 to 6, transformed cell culture, root, and leaf.

    Article Snippet: Hybridizing peptides were detected using horseradish peroxidase-linked secondary antibody, with Lumi-Phos WB chemiluminescent reagent (Pierce Chemical, Rockford, Illinois).

    Techniques: Activity Assay, Expressing, Transgenic Assay, Transformation Assay, Western Blot, Cell Culture

    HDAC11 knockdown affects acetylation levels of histone H3 in T. castaneum . (A) The total protein extracted from dsHDAC11 or dsmalE injected larvae were resolved on SDS-PAGE gels, transferred to western blots, and probed with antibodies recognizing Acetyl-Histone H3 (Antibody Sampler Kit # 9927-Cell Signaling (H3K9, H3K14, H3K18, H3K27, and H3K56). ß-actin served as a loading control. The HRP-linked IgG (#7074-Cell Signaling) was used as a secondary antibody. Band densities were determined by Image-J software and normalized with loading control protein-ß-Actin. The Mean + SE ( n = 3) band densities are shown. Means marked with different letters are significantly different from each other, P ≤ 0.05 by ANOVA. (B) Acetylated-Lysine (Ac-K 2 -100) MultiMab TM Rabbit mAb mix #9814 was used to detect acetylation levels of proteins extracted from TcA cells exposed to dsHDAC11 or dsmalE. The acetylation levels of histone H3K9, H3K18 and H3K27 increased in HDAC11 knockdown cells were detected as described in Figure 7A . The band densities were quantified and plotted as described in Figure 7A . The Mean + SE ( n = 3) band densities are shown. Means marked with different letters are significantly different from each other, P ≤ 0.05 by ANOVA.

    Journal: Frontiers in Genetics

    Article Title: Histone Deacetylase 11 Knockdown Blocks Larval Development and Metamorphosis in the Red Flour Beetle, Tribolium castaneum

    doi: 10.3389/fgene.2020.00683

    Figure Lengend Snippet: HDAC11 knockdown affects acetylation levels of histone H3 in T. castaneum . (A) The total protein extracted from dsHDAC11 or dsmalE injected larvae were resolved on SDS-PAGE gels, transferred to western blots, and probed with antibodies recognizing Acetyl-Histone H3 (Antibody Sampler Kit # 9927-Cell Signaling (H3K9, H3K14, H3K18, H3K27, and H3K56). ß-actin served as a loading control. The HRP-linked IgG (#7074-Cell Signaling) was used as a secondary antibody. Band densities were determined by Image-J software and normalized with loading control protein-ß-Actin. The Mean + SE ( n = 3) band densities are shown. Means marked with different letters are significantly different from each other, P ≤ 0.05 by ANOVA. (B) Acetylated-Lysine (Ac-K 2 -100) MultiMab TM Rabbit mAb mix #9814 was used to detect acetylation levels of proteins extracted from TcA cells exposed to dsHDAC11 or dsmalE. The acetylation levels of histone H3K9, H3K18 and H3K27 increased in HDAC11 knockdown cells were detected as described in Figure 7A . The band densities were quantified and plotted as described in Figure 7A . The Mean + SE ( n = 3) band densities are shown. Means marked with different letters are significantly different from each other, P ≤ 0.05 by ANOVA.

    Article Snippet: The Anti-rabbit IgG, HRP-linked antibody (Cell signaling #7074), was used for chemiluminescence detection.

    Techniques: Injection, SDS Page, Western Blot, Software

    PRX-1 promotes phosphorylation of STAT3. Whole protein was extracted through RIPA buffer and after performing SDS-PAGE electrophoresis, the protein was transferred to a PVDF membrane. The membrane was subsequently blocked and blots were visualized using antibodies for β-actin, phosphor-STAT3 and STAT3 and anti-mouse IgG or anti-rabbit IgG HRP-linked antibodies.

    Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

    Article Title: Effect of PRX-1 Downregulation in the Type 1 Diabetes Microenvironment

    doi: 10.4196/kjpp.2012.16.6.463

    Figure Lengend Snippet: PRX-1 promotes phosphorylation of STAT3. Whole protein was extracted through RIPA buffer and after performing SDS-PAGE electrophoresis, the protein was transferred to a PVDF membrane. The membrane was subsequently blocked and blots were visualized using antibodies for β-actin, phosphor-STAT3 and STAT3 and anti-mouse IgG or anti-rabbit IgG HRP-linked antibodies.

    Article Snippet: The membranes were then incubated with anti-mouse IgG or anti-rabbit IgG HRP-linked antibodies (Cell Signaling), and developed using the ChemiGlow West (Alpha Innotech).

    Techniques: SDS Page, Electrophoresis

    CA192-CsA prevents NFAT1 and NFAT2 dephosphorylation. 10 μg of total protein in the supernatant of cell lysate was resolve in each lane by SDS-PAGE. Proteins on gels were transferred to membrane and labeled with anti-NFAT1 or 2 primary rabbit antibodies followed by anti-rabbit IgG, HRP-linked secondary antibody. Jurkat cell pretreatments were with CA192-CsA or with free CsA (dissolved in 2.5% v/v DMSO) at 1 μM CsA concentration, or with vehicle controls, prior to stimulation with 20 ng/mL PMA and 1 μg/mL ionomycin. CA192-CsA and free CsA treatments showed retention of NFAT1 and 2 at their phosphorylated forms with stimulation but neither CA192 vehicle nor DMSO control (2.5%v/v) prevented NFAT dephosphorylation upon stimulation. The cell lysis method used here was not optimized for nuclear protein extraction, which explains why the bands of dephosphorylated NFATs have lower intensity associated with nuclear translocation. The blots shown here are representatives of three independent repeats.

    Journal: Journal of controlled release : official journal of the Controlled Release Society

    Article Title: A novel elastin-like polypeptide drug carrier for cyclosporine A improves tear flow in a mouse model of Sjögren’s Syndrome

    doi: 10.1016/j.jconrel.2018.10.026

    Figure Lengend Snippet: CA192-CsA prevents NFAT1 and NFAT2 dephosphorylation. 10 μg of total protein in the supernatant of cell lysate was resolve in each lane by SDS-PAGE. Proteins on gels were transferred to membrane and labeled with anti-NFAT1 or 2 primary rabbit antibodies followed by anti-rabbit IgG, HRP-linked secondary antibody. Jurkat cell pretreatments were with CA192-CsA or with free CsA (dissolved in 2.5% v/v DMSO) at 1 μM CsA concentration, or with vehicle controls, prior to stimulation with 20 ng/mL PMA and 1 μg/mL ionomycin. CA192-CsA and free CsA treatments showed retention of NFAT1 and 2 at their phosphorylated forms with stimulation but neither CA192 vehicle nor DMSO control (2.5%v/v) prevented NFAT dephosphorylation upon stimulation. The cell lysis method used here was not optimized for nuclear protein extraction, which explains why the bands of dephosphorylated NFATs have lower intensity associated with nuclear translocation. The blots shown here are representatives of three independent repeats.

    Article Snippet: NFAT1 primary antibody (4389) and NFAT2 (8032) primary antibody and anti-rabbit IgG, HRP-linked secondary antibody (7074) were generated by Cell Signaling Technology (Danvers, MA).

    Techniques: De-Phosphorylation Assay, SDS Page, Labeling, Concentration Assay, Lysis, Protein Extraction, Translocation Assay

    Muscle damage markers in serum and inflammation in gastrocnemius muscle are decreased in 3-month-old dystrophic mice lacking miR-378. ( A ) Lower serum activity of LDH in mdx animals lacking miR-378; activity assay; n = 10–14/group. ( B ) Increased serum CK activity in mdx mice with a tendency to be decreased by miR-378-KO, activity assay; n = 11–13/group. ( C ) Necrosis assessment by immunofluorescent staining of IgM and IgG (green) binding and its calculation indicating no differences between groups; n = 9–10/group. Scale bar: 100 μm. ( D ) Representative pictures of H E staining of gastrocnemius muscle with semiquantitative analysis of inflammation extent showing a tendency in decreased inflammatory cell infiltration in dKO mice; microscopic assessment using Nikon Eclipse microscope. Scale bar: 100 μm; n = 4–6/group. ( E ) Decreased number of WBC in the peripheral blood in dKO mice; blood cell count; n = 5–6/group. ( F–J ) The analysis of inflammatory cells in hind limb muscles with special emphasis on macrophage subpopulations; flow cytometry analysis calculated as the percentage of Hoechst + cells; n = 5/group. The percentage of CD45 + cells ( F ), macrophages (CD45 + F4/80 + CD11b + cells) ( G ), M1-like macrophages (CD45 + F4/80 + CD11b + MHCII hi CD206 lo cells) ( H ), M2-like macrophages (CD45 + F4/80 + CD11b + MHCII lo CD206 hi cells) ( I ), and eosinophils (CD45 + F4/80 + CD86 + cells) ( J ) showing significant decrease in dKO mice. ( K ) The decreased HO-1 protein level in dKO animals assessed by Western blot; GAPDH used as loading control. Representative result of 2 independent experiments; n = 4–5/group. Data are presented as mean ± SEM. * P

    Journal: JCI Insight

    Article Title: Lack of miR-378 attenuates muscular dystrophy in mdx mice

    doi: 10.1172/jci.insight.135576

    Figure Lengend Snippet: Muscle damage markers in serum and inflammation in gastrocnemius muscle are decreased in 3-month-old dystrophic mice lacking miR-378. ( A ) Lower serum activity of LDH in mdx animals lacking miR-378; activity assay; n = 10–14/group. ( B ) Increased serum CK activity in mdx mice with a tendency to be decreased by miR-378-KO, activity assay; n = 11–13/group. ( C ) Necrosis assessment by immunofluorescent staining of IgM and IgG (green) binding and its calculation indicating no differences between groups; n = 9–10/group. Scale bar: 100 μm. ( D ) Representative pictures of H E staining of gastrocnemius muscle with semiquantitative analysis of inflammation extent showing a tendency in decreased inflammatory cell infiltration in dKO mice; microscopic assessment using Nikon Eclipse microscope. Scale bar: 100 μm; n = 4–6/group. ( E ) Decreased number of WBC in the peripheral blood in dKO mice; blood cell count; n = 5–6/group. ( F–J ) The analysis of inflammatory cells in hind limb muscles with special emphasis on macrophage subpopulations; flow cytometry analysis calculated as the percentage of Hoechst + cells; n = 5/group. The percentage of CD45 + cells ( F ), macrophages (CD45 + F4/80 + CD11b + cells) ( G ), M1-like macrophages (CD45 + F4/80 + CD11b + MHCII hi CD206 lo cells) ( H ), M2-like macrophages (CD45 + F4/80 + CD11b + MHCII lo CD206 hi cells) ( I ), and eosinophils (CD45 + F4/80 + CD86 + cells) ( J ) showing significant decrease in dKO mice. ( K ) The decreased HO-1 protein level in dKO animals assessed by Western blot; GAPDH used as loading control. Representative result of 2 independent experiments; n = 4–5/group. Data are presented as mean ± SEM. * P

    Article Snippet: Primary antibodies — mouse monoclonal anti-FGF1 (AF4686, R & D Systems), mouse monoclonal anti-GAPDH (sc-59540, Santa Cruz Biotechnology Inc.), rabbit polyclonal anti–HO-1 (ADI-SPA-894, Enzo Life Sciences), rabbit polyclonal anti-AMPKA (2603S, Cell Signalling Technology), and rabbit monoclonal anti–PGC1-β (ab176328, Abcam) — and secondary antibodies (conjugated with HRP) include anti–mouse Ig (554002, BD Biosciences) for the detection of GAPDH or FGF1, as well as anti–rabbit IgG (7074, Cell Signaling Technology) for the detection of HO-1, AMPKA, PGC1-β, were used.

    Techniques: Mouse Assay, Activity Assay, Staining, Binding Assay, Microscopy, Cell Counting, Flow Cytometry, Western Blot

    Interaction of AOPPs-HSA with RAGE: role on AOPPs-HSA-induced ROS generation. ( A ) Microplate wells were coated with 10 μ g/ml of AOPPs-HSA, CML-HSA, GA-HSA, or native HSA. After washing and blocking, the wells were incubated with indicated concentrations of sRAGE. The bound sRAGE was detected with a HRP-conjugated monoclonal α -RAGE IgG. ( B ) CML-HSA (10 μ g/ml) was immobilized onto the wells. sRAGE (4 μ g/ml) was pre-incubated with indicated concentrations of AOPPs-HSA, CML-HSA, GA-HSA, or native HSA, and then added to the wells. The binding of sRAGE with immobilized AGEs-HSA was detected as described in A . ( C ) HUVECs were pre-incubated with a polyclonal α -RAGE IgG, a nonimmune IgG, or excess sRAGE, and then stimulated with AOPPs-HSA. ROS generation was determined by measuring the DCF fluorescence. ( D ) AOPPs-HSA (200 μ g/ml) was pre-incubated with indicated concentrations of an α -AOPPs or a nonimmune IgG, and then interacted with HUVECs. ROS generation was determined as described above ( lower panel ). The α -AOPPs recognized AOPPs-HSA in Western blot ( upper panel, lane 1 ), but did not react with CML- ( lane 2 ), GA- ( lane 3 ), GC- ( lane 4 ), and RB-derived AGEs ( lane 5 ) or native HSA ( lane 6 ). ( E ) AOPPs-HSA was pre-incubated with indicated concentrations of 6D12 antibody and then interacted with HUVECs. ROS generation was measured as described above. ( F ) 6D12 antibody reacted with CML, GA- and GC-derived AGEs, and, to a lesser extent, with RB-derived protein. Data from three independent experiments are shown as mean ± SEM. ANOVA, p

    Journal: Antioxidants & Redox Signaling

    Article Title: Advanced Oxidation Protein Products Activate Vascular Endothelial Cells via a RAGE-Mediated Signaling Pathway

    doi: 10.1089/ars.2007.1999

    Figure Lengend Snippet: Interaction of AOPPs-HSA with RAGE: role on AOPPs-HSA-induced ROS generation. ( A ) Microplate wells were coated with 10 μ g/ml of AOPPs-HSA, CML-HSA, GA-HSA, or native HSA. After washing and blocking, the wells were incubated with indicated concentrations of sRAGE. The bound sRAGE was detected with a HRP-conjugated monoclonal α -RAGE IgG. ( B ) CML-HSA (10 μ g/ml) was immobilized onto the wells. sRAGE (4 μ g/ml) was pre-incubated with indicated concentrations of AOPPs-HSA, CML-HSA, GA-HSA, or native HSA, and then added to the wells. The binding of sRAGE with immobilized AGEs-HSA was detected as described in A . ( C ) HUVECs were pre-incubated with a polyclonal α -RAGE IgG, a nonimmune IgG, or excess sRAGE, and then stimulated with AOPPs-HSA. ROS generation was determined by measuring the DCF fluorescence. ( D ) AOPPs-HSA (200 μ g/ml) was pre-incubated with indicated concentrations of an α -AOPPs or a nonimmune IgG, and then interacted with HUVECs. ROS generation was determined as described above ( lower panel ). The α -AOPPs recognized AOPPs-HSA in Western blot ( upper panel, lane 1 ), but did not react with CML- ( lane 2 ), GA- ( lane 3 ), GC- ( lane 4 ), and RB-derived AGEs ( lane 5 ) or native HSA ( lane 6 ). ( E ) AOPPs-HSA was pre-incubated with indicated concentrations of 6D12 antibody and then interacted with HUVECs. ROS generation was measured as described above. ( F ) 6D12 antibody reacted with CML, GA- and GC-derived AGEs, and, to a lesser extent, with RB-derived protein. Data from three independent experiments are shown as mean ± SEM. ANOVA, p

    Article Snippet: Cell homogenates (40 μ g protein in each sample) were immunoblotted using the rabbit polyclonal antibodies against human pan- or phospho-ERK1/2, p38MAPK , and JNK1/2 as the primary antibodies, and a HRP-linked anti-rabbit IgG as the secondary antibody (all from Cell Signaling Technology).

    Techniques: Blocking Assay, Incubation, Binding Assay, Fluorescence, Western Blot, Derivative Assay

    Protein phosphorylation associated with myometrial contractility. Myometrial strips were snap frozen at specific stages during the development of contractions. The tissue was then pulverised, protein extracted and separated by 1D SDS-PAGE. (A) Phosphorylated myometrial proteins were labelled in gel with Pro-Q® Diamond Phospho Stain and visualised using a FujiFilm LAS-3000 luminescent image analyser. The gel was then SYPRO® Ruby Protein stained to visualise total protein and ascertain equal loading. Lanes M P and M T illustrate the Peppermint™ Stick molecular weight marker labelled with Pro-Q® Diamond Stain and SYPRO Ruby Protein Stain respectively. Dotted line indicates removal on none-relevant gel lanes. (B) Separated proteins were western transferred to nitrocellulose membrane. Total protein loading was visualised using SYPRO Ruby Blot Stain and equal loading across lanes confirmed. Membranes were then probed with mouse anti-phosphotyrosine (1∶2500) and anti-mouse IgG-HRP (1∶2500). The immunoreactive bands were detected using a FujiFilm LAS-3000 luminescent image analyser. Positive and negative controls were protein extracts from capacitated and non-capacitated human spermatozoa respectively. Black arrowheads and white arrowheads indicate phosphorylation events associated with contraction and relaxation respectively. (i), (ii) and (iv) highlight bands exhibiting phasic phosphorylation, whilst phosphorylation remained increased for bands (iii) and (v). Representative images shown from 3 replicates. M r = relative molecular mass×1000. M T = molecular weight marker stained for total protein detection, M P = molecular weight marker stained for phospho protein detection.

    Journal: PLoS ONE

    Article Title: Phasic Phosphorylation of Caldesmon and ERK 1/2 during Contractions in Human Myometrium

    doi: 10.1371/journal.pone.0021542

    Figure Lengend Snippet: Protein phosphorylation associated with myometrial contractility. Myometrial strips were snap frozen at specific stages during the development of contractions. The tissue was then pulverised, protein extracted and separated by 1D SDS-PAGE. (A) Phosphorylated myometrial proteins were labelled in gel with Pro-Q® Diamond Phospho Stain and visualised using a FujiFilm LAS-3000 luminescent image analyser. The gel was then SYPRO® Ruby Protein stained to visualise total protein and ascertain equal loading. Lanes M P and M T illustrate the Peppermint™ Stick molecular weight marker labelled with Pro-Q® Diamond Stain and SYPRO Ruby Protein Stain respectively. Dotted line indicates removal on none-relevant gel lanes. (B) Separated proteins were western transferred to nitrocellulose membrane. Total protein loading was visualised using SYPRO Ruby Blot Stain and equal loading across lanes confirmed. Membranes were then probed with mouse anti-phosphotyrosine (1∶2500) and anti-mouse IgG-HRP (1∶2500). The immunoreactive bands were detected using a FujiFilm LAS-3000 luminescent image analyser. Positive and negative controls were protein extracts from capacitated and non-capacitated human spermatozoa respectively. Black arrowheads and white arrowheads indicate phosphorylation events associated with contraction and relaxation respectively. (i), (ii) and (iv) highlight bands exhibiting phasic phosphorylation, whilst phosphorylation remained increased for bands (iii) and (v). Representative images shown from 3 replicates. M r = relative molecular mass×1000. M T = molecular weight marker stained for total protein detection, M P = molecular weight marker stained for phospho protein detection.

    Article Snippet: Washed blots were then incubated in horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (cat.# 7074, Cell Signaling) or anti-mouse IgG (cat.# 7076, Cell Signaling) secondary antibody as appropriate.

    Techniques: SDS Page, Staining, Molecular Weight, Marker, Western Blot

    Binding of MBP–Sbi 41–436 , MBP–Sbi 41–253 and MBP–Sbi 254–436 to whole cells of Newman Spa - Sbi - . A. Binding of MBP–Sbi 41–436 , MBP–Sbi 254–436 MBP–Sbi 41–253 and MBP to wells coated with whole Newman Spa - Sbi - cells. Recombinant protein binding was detected with HRP-conjugated anti-MBP IgG. Binding assay was preformed n = 3 times with similar results. The graph shown is a representative of one experiment with each plot in the graph representing the average of duplicate wells. B. Washed whole cells of Newman Spa - Sbi - were incubated with 0.5 µM MBP–Sbi 41–436 , MBP–Sbi 254–436, MBP–Sbi 41–253 and MBP followed by mouse anti-MBP antiserum and FITC-labelled rabbit anti-mouse IgG. Fluorescence intensity was measured by flow cytometry. The assay was preformed n = 3 times. Each plot on the graph represents the average value for all three replicas. Error bars show the standard deviation. C. A representative flow cytometry trace of recombinant Sbi derivatives binding to Newman Spa - Sbi - . D. Recombinant MBP–Sbi 41–436 , MBP–Sbi 254–436 MBP–Sbi 41–253 and MBP were incubated with whole cells of Newman Spa - Sbi - and fractionated. Cytoplasmic membrane fractions were analysed by Western blotting with HRP-conjugated anti-MBP IgG or rabbit anti-EbpS IgG followed by HRP-conjugated protein A. All immunoblotting experiments were repeated n = 3 times.

    Journal: Molecular Microbiology

    Article Title: The immune evasion protein Sbi of Staphylococcus aureus occurs both extracellularly and anchored to the cell envelope by binding lipoteichoic acid

    doi: 10.1111/j.1365-2958.2011.07966.x

    Figure Lengend Snippet: Binding of MBP–Sbi 41–436 , MBP–Sbi 41–253 and MBP–Sbi 254–436 to whole cells of Newman Spa - Sbi - . A. Binding of MBP–Sbi 41–436 , MBP–Sbi 254–436 MBP–Sbi 41–253 and MBP to wells coated with whole Newman Spa - Sbi - cells. Recombinant protein binding was detected with HRP-conjugated anti-MBP IgG. Binding assay was preformed n = 3 times with similar results. The graph shown is a representative of one experiment with each plot in the graph representing the average of duplicate wells. B. Washed whole cells of Newman Spa - Sbi - were incubated with 0.5 µM MBP–Sbi 41–436 , MBP–Sbi 254–436, MBP–Sbi 41–253 and MBP followed by mouse anti-MBP antiserum and FITC-labelled rabbit anti-mouse IgG. Fluorescence intensity was measured by flow cytometry. The assay was preformed n = 3 times. Each plot on the graph represents the average value for all three replicas. Error bars show the standard deviation. C. A representative flow cytometry trace of recombinant Sbi derivatives binding to Newman Spa - Sbi - . D. Recombinant MBP–Sbi 41–436 , MBP–Sbi 254–436 MBP–Sbi 41–253 and MBP were incubated with whole cells of Newman Spa - Sbi - and fractionated. Cytoplasmic membrane fractions were analysed by Western blotting with HRP-conjugated anti-MBP IgG or rabbit anti-EbpS IgG followed by HRP-conjugated protein A. All immunoblotting experiments were repeated n = 3 times.

    Article Snippet: Bound LTA was detected with an LTA (polyglycerolphosphate)-specific monoclonal antibody (clone 55, HyCult Biotechnology) followed by HRP-linked anti-mouse IgG (Cell Signalling).

    Techniques: Binding Assay, Recombinant, Protein Binding, Incubation, Fluorescence, Flow Cytometry, Cytometry, Standard Deviation, Western Blot

    Binding of MBP–Sbi 303–436 to the cytoplasmic membranes of Gram-positive bacteria. A. Cytoplasmic membrane material purified from S. aureus , S. epidermidis , S. lugdunensis , L. monocytogenes and E. coli were incubated with MBP–Sbi 303–436 and MBP in microtitre plates. Recombinant protein binding was detected with HRP-conjugated anti-MBP IgG. B. Recombinant MBP–Sbi 303–436 was pre-incubated with increasing concentrations of either S. aureus LTA or bovine serum, albumin (BSA) (0–200 µg ml −1 ) before being added to cytoplasmic membrane-coated microtitre plates. Recombinant protein binding was detected with HRP-conjugated anti-MBP IgG. Each assay was preformed n = 3 times with similar results. The graphs shown are representatives of one experiment with each plot in the graph representing the average of duplicate wells.

    Journal: Molecular Microbiology

    Article Title: The immune evasion protein Sbi of Staphylococcus aureus occurs both extracellularly and anchored to the cell envelope by binding lipoteichoic acid

    doi: 10.1111/j.1365-2958.2011.07966.x

    Figure Lengend Snippet: Binding of MBP–Sbi 303–436 to the cytoplasmic membranes of Gram-positive bacteria. A. Cytoplasmic membrane material purified from S. aureus , S. epidermidis , S. lugdunensis , L. monocytogenes and E. coli were incubated with MBP–Sbi 303–436 and MBP in microtitre plates. Recombinant protein binding was detected with HRP-conjugated anti-MBP IgG. B. Recombinant MBP–Sbi 303–436 was pre-incubated with increasing concentrations of either S. aureus LTA or bovine serum, albumin (BSA) (0–200 µg ml −1 ) before being added to cytoplasmic membrane-coated microtitre plates. Recombinant protein binding was detected with HRP-conjugated anti-MBP IgG. Each assay was preformed n = 3 times with similar results. The graphs shown are representatives of one experiment with each plot in the graph representing the average of duplicate wells.

    Article Snippet: Bound LTA was detected with an LTA (polyglycerolphosphate)-specific monoclonal antibody (clone 55, HyCult Biotechnology) followed by HRP-linked anti-mouse IgG (Cell Signalling).

    Techniques: Binding Assay, Purification, Incubation, Recombinant, Protein Binding

    Binding of MBP–Sbi to whole cells and purified cytoplasmic membrane. Binding of MBP–Sbi 41–436, MBP–Sbi 254–436 MBP–Sbi 41–253 and MBP to wells coated with (A) S. aureus Newman Spa - Sbi + cells, (B) Newman Spa - Sbi - cells, (C) Newman Spa - Sbi + cytoplasmic membrane material and (D) Newman Spa - Sbi - cytoplasmic membrane material. Closed symbols and black lines refer to Newman Spa - . Recombinant protein binding was detected with HRP-conjugated anti-MBP IgG. Each assay was preformed n = 3 times with similar results. The graphs shown are representatives of one experiment with each plot in the graph representing the average of duplicate wells. Blots shown are representative of three independent experiments.

    Journal: Molecular Microbiology

    Article Title: The immune evasion protein Sbi of Staphylococcus aureus occurs both extracellularly and anchored to the cell envelope by binding lipoteichoic acid

    doi: 10.1111/j.1365-2958.2011.07966.x

    Figure Lengend Snippet: Binding of MBP–Sbi to whole cells and purified cytoplasmic membrane. Binding of MBP–Sbi 41–436, MBP–Sbi 254–436 MBP–Sbi 41–253 and MBP to wells coated with (A) S. aureus Newman Spa - Sbi + cells, (B) Newman Spa - Sbi - cells, (C) Newman Spa - Sbi + cytoplasmic membrane material and (D) Newman Spa - Sbi - cytoplasmic membrane material. Closed symbols and black lines refer to Newman Spa - . Recombinant protein binding was detected with HRP-conjugated anti-MBP IgG. Each assay was preformed n = 3 times with similar results. The graphs shown are representatives of one experiment with each plot in the graph representing the average of duplicate wells. Blots shown are representative of three independent experiments.

    Article Snippet: Bound LTA was detected with an LTA (polyglycerolphosphate)-specific monoclonal antibody (clone 55, HyCult Biotechnology) followed by HRP-linked anti-mouse IgG (Cell Signalling).

    Techniques: Binding Assay, Purification, Recombinant, Protein Binding

    Analysis of the Sbi–LTA interaction. A. Displacement of Sbi from the cytoplasmic membrane by soluble LTA. Newman Spa - cytoplasmic membrane was incubated with increasing amounts of S. aureus LTA (0–400 µg ml −1 ). Sbi bound to the cytoplasmic membrane or released into the supernatant was detected using rabbit anti-Sbi D3D4WrY IgG followed by HRP-conjugated goat anti-rabbit IgG. B. Far Western blotting of S. aureus cytoplasmic membrane fractions with LTA. Newman Spa - Sbi - and Newman Spa - Sbi - (pRMC2- sbi Δ D1D2 ) cytoplasmic membrane material was fractionated by SDS-PAGE, transferred to a nitrocellulose membrane incubated with purified LTA and bound LTA was detected with anti-LTA monoclonal antibody followed by HRP-linked rabbit anti-mouse IgG. All immunoblotting experiments were repeated n = 3 times.

    Journal: Molecular Microbiology

    Article Title: The immune evasion protein Sbi of Staphylococcus aureus occurs both extracellularly and anchored to the cell envelope by binding lipoteichoic acid

    doi: 10.1111/j.1365-2958.2011.07966.x

    Figure Lengend Snippet: Analysis of the Sbi–LTA interaction. A. Displacement of Sbi from the cytoplasmic membrane by soluble LTA. Newman Spa - cytoplasmic membrane was incubated with increasing amounts of S. aureus LTA (0–400 µg ml −1 ). Sbi bound to the cytoplasmic membrane or released into the supernatant was detected using rabbit anti-Sbi D3D4WrY IgG followed by HRP-conjugated goat anti-rabbit IgG. B. Far Western blotting of S. aureus cytoplasmic membrane fractions with LTA. Newman Spa - Sbi - and Newman Spa - Sbi - (pRMC2- sbi Δ D1D2 ) cytoplasmic membrane material was fractionated by SDS-PAGE, transferred to a nitrocellulose membrane incubated with purified LTA and bound LTA was detected with anti-LTA monoclonal antibody followed by HRP-linked rabbit anti-mouse IgG. All immunoblotting experiments were repeated n = 3 times.

    Article Snippet: Bound LTA was detected with an LTA (polyglycerolphosphate)-specific monoclonal antibody (clone 55, HyCult Biotechnology) followed by HRP-linked anti-mouse IgG (Cell Signalling).

    Techniques: Incubation, Far Western Blot, SDS Page, Purification

    Cellular localization of Sbi C-terminal truncates. A. Coomassie stain of an SDS-PAGE gel of MBP–Sbi 303–436 . Western immunoblot of MBP–Sbi 303–436 probed with HRP-conjugated anti-MBP IgG. B. Binding of MBP–Sbi 303–436 and MBP to plates coated in Newman Spa - Sbi - cytoplasmic membrane material. C. Binding of MBP–Sbi 254–436 , MBP–Sbi 303–436 and MBP to Newman Spa - Sbi - LTA-coated wells. Recombinant protein binding was detected with HRP-conjugated anti-MBP IgG. Each assay was preformed n = 3 times with similar results. The graphs shown are representatives of one experiment with each plot in the graph representing the average of duplicate wells. D. Cytoplasmic membrane and culture supernatant fractions of Newman Spa - Sbi - (pRMC2- sbi ) C-terminal truncates analysed by Western immunoblotting with HRP-labelled rabbit IgG.

    Journal: Molecular Microbiology

    Article Title: The immune evasion protein Sbi of Staphylococcus aureus occurs both extracellularly and anchored to the cell envelope by binding lipoteichoic acid

    doi: 10.1111/j.1365-2958.2011.07966.x

    Figure Lengend Snippet: Cellular localization of Sbi C-terminal truncates. A. Coomassie stain of an SDS-PAGE gel of MBP–Sbi 303–436 . Western immunoblot of MBP–Sbi 303–436 probed with HRP-conjugated anti-MBP IgG. B. Binding of MBP–Sbi 303–436 and MBP to plates coated in Newman Spa - Sbi - cytoplasmic membrane material. C. Binding of MBP–Sbi 254–436 , MBP–Sbi 303–436 and MBP to Newman Spa - Sbi - LTA-coated wells. Recombinant protein binding was detected with HRP-conjugated anti-MBP IgG. Each assay was preformed n = 3 times with similar results. The graphs shown are representatives of one experiment with each plot in the graph representing the average of duplicate wells. D. Cytoplasmic membrane and culture supernatant fractions of Newman Spa - Sbi - (pRMC2- sbi ) C-terminal truncates analysed by Western immunoblotting with HRP-labelled rabbit IgG.

    Article Snippet: Bound LTA was detected with an LTA (polyglycerolphosphate)-specific monoclonal antibody (clone 55, HyCult Biotechnology) followed by HRP-linked anti-mouse IgG (Cell Signalling).

    Techniques: Staining, SDS Page, Western Blot, Binding Assay, Recombinant, Protein Binding

    Interaction of MBP–Sbi with LTA. A. Binding of MBP–Sbi 41–436 , MBP–Sbi 254–436 MBP–Sbi 41–253 and MBP to LTA-coated wells. B and C. (B) Inhibition of binding of MBP–Sbi 41–436 and MBP–Sbi 254–436 to LTA and (C) to the purified cytoplasmic membrane fraction from S. aureus Newman Spa - Sbi - . Recombinant proteins were pre-incubated with increasing concentrations of either S. aureus LTA or heparin sulphate (0–200 µg ml −1 ) before being added to coated microtitre plates. D. Binding of MBP–Sbi proteins to purified cell wall-coated wells. In each assay recombinant protein binding was detected with HRP-conjugated anti-MBP IgG. Each assay was preformed n = 3 times with similar results. The graphs shown are representatives of one experiment with each plot in the graph representing the average of triplicate wells.

    Journal: Molecular Microbiology

    Article Title: The immune evasion protein Sbi of Staphylococcus aureus occurs both extracellularly and anchored to the cell envelope by binding lipoteichoic acid

    doi: 10.1111/j.1365-2958.2011.07966.x

    Figure Lengend Snippet: Interaction of MBP–Sbi with LTA. A. Binding of MBP–Sbi 41–436 , MBP–Sbi 254–436 MBP–Sbi 41–253 and MBP to LTA-coated wells. B and C. (B) Inhibition of binding of MBP–Sbi 41–436 and MBP–Sbi 254–436 to LTA and (C) to the purified cytoplasmic membrane fraction from S. aureus Newman Spa - Sbi - . Recombinant proteins were pre-incubated with increasing concentrations of either S. aureus LTA or heparin sulphate (0–200 µg ml −1 ) before being added to coated microtitre plates. D. Binding of MBP–Sbi proteins to purified cell wall-coated wells. In each assay recombinant protein binding was detected with HRP-conjugated anti-MBP IgG. Each assay was preformed n = 3 times with similar results. The graphs shown are representatives of one experiment with each plot in the graph representing the average of triplicate wells.

    Article Snippet: Bound LTA was detected with an LTA (polyglycerolphosphate)-specific monoclonal antibody (clone 55, HyCult Biotechnology) followed by HRP-linked anti-mouse IgG (Cell Signalling).

    Techniques: Binding Assay, Inhibition, Purification, Recombinant, Incubation, Protein Binding

    Sbi cellular location in LTA-negative strains. A. Whole-cell lysate fractions of RN4220 Spa - , 4S5 and 4S5 (pCN34- ltaS ) analysed by Western immunoblotting with monoclonal mouse anti-LTA antibodies followed by HRP-conjugated rabbit anti-mouse IgG. B. Cytoplasmic membrane and culture supernatant fractions of RN4220 Spa - , 4S5 and 4S5 (pCN34- ltaS ) analysed by Western immunoblotting with rabbit anti-Sbi D3D4WrY IgG and HRP-conjugated goat anti-rabbit IgG (i and ii) and rabbit anti-EbpS IgG followed by HRP-conjugated goat anti-rabbit IgG (iii). Blots shown are representative of three independent experiments. Densitometric analysis was carried out using ImageJ software. Integrated band densities were measured with correction of background. Values given are the mean ± the standard deviation of n = 3 experiments

    Journal: Molecular Microbiology

    Article Title: The immune evasion protein Sbi of Staphylococcus aureus occurs both extracellularly and anchored to the cell envelope by binding lipoteichoic acid

    doi: 10.1111/j.1365-2958.2011.07966.x

    Figure Lengend Snippet: Sbi cellular location in LTA-negative strains. A. Whole-cell lysate fractions of RN4220 Spa - , 4S5 and 4S5 (pCN34- ltaS ) analysed by Western immunoblotting with monoclonal mouse anti-LTA antibodies followed by HRP-conjugated rabbit anti-mouse IgG. B. Cytoplasmic membrane and culture supernatant fractions of RN4220 Spa - , 4S5 and 4S5 (pCN34- ltaS ) analysed by Western immunoblotting with rabbit anti-Sbi D3D4WrY IgG and HRP-conjugated goat anti-rabbit IgG (i and ii) and rabbit anti-EbpS IgG followed by HRP-conjugated goat anti-rabbit IgG (iii). Blots shown are representative of three independent experiments. Densitometric analysis was carried out using ImageJ software. Integrated band densities were measured with correction of background. Values given are the mean ± the standard deviation of n = 3 experiments

    Article Snippet: Bound LTA was detected with an LTA (polyglycerolphosphate)-specific monoclonal antibody (clone 55, HyCult Biotechnology) followed by HRP-linked anti-mouse IgG (Cell Signalling).

    Techniques: Western Blot, Software, Standard Deviation

    Binding of MBP–Sbi 41–436 , MBP–Sbi 41–253 and MBP–Sbi 254–436 to purified cytoplasmic membrane. A. Schematic diagram of Sbi showing the residues present in each recombinant MBP-tagged protein. B. Coomassie stain of an SDS-PAGE gel of MBP–Sbi recombinant proteins. C. Western immunoblot of MBP–Sbi recombinant proteins probed with HRP-conjugated anti-MBP IgG. D. Binding of MBP–Sbi 41–436 , MBP–Sbi 254–436 MBP–Sbi 41–253 and MBP to wells coated with cytoplasmic membrane material isolated from Newman Spa - Sbi - cells. Recombinant protein binding was detected with HRP-conjugated anti-MBP IgG. Binding assay was preformed n = 3 times with similar results. The graph shown is a representative of one experiment with each point plot representing the average of duplicate wells.

    Journal: Molecular Microbiology

    Article Title: The immune evasion protein Sbi of Staphylococcus aureus occurs both extracellularly and anchored to the cell envelope by binding lipoteichoic acid

    doi: 10.1111/j.1365-2958.2011.07966.x

    Figure Lengend Snippet: Binding of MBP–Sbi 41–436 , MBP–Sbi 41–253 and MBP–Sbi 254–436 to purified cytoplasmic membrane. A. Schematic diagram of Sbi showing the residues present in each recombinant MBP-tagged protein. B. Coomassie stain of an SDS-PAGE gel of MBP–Sbi recombinant proteins. C. Western immunoblot of MBP–Sbi recombinant proteins probed with HRP-conjugated anti-MBP IgG. D. Binding of MBP–Sbi 41–436 , MBP–Sbi 254–436 MBP–Sbi 41–253 and MBP to wells coated with cytoplasmic membrane material isolated from Newman Spa - Sbi - cells. Recombinant protein binding was detected with HRP-conjugated anti-MBP IgG. Binding assay was preformed n = 3 times with similar results. The graph shown is a representative of one experiment with each point plot representing the average of duplicate wells.

    Article Snippet: Bound LTA was detected with an LTA (polyglycerolphosphate)-specific monoclonal antibody (clone 55, HyCult Biotechnology) followed by HRP-linked anti-mouse IgG (Cell Signalling).

    Techniques: Binding Assay, Purification, Recombinant, Staining, SDS Page, Western Blot, Isolation, Protein Binding

    Surface expression of Sbi domains D3D4. Serial dilutions of cells were applied to a nitrocellulose membrane and probed with rabbit anti-Sbi D3D4WrY IgG followed by HRP-conjugated goat anti-rabbit IgG.

    Journal: Molecular Microbiology

    Article Title: The immune evasion protein Sbi of Staphylococcus aureus occurs both extracellularly and anchored to the cell envelope by binding lipoteichoic acid

    doi: 10.1111/j.1365-2958.2011.07966.x

    Figure Lengend Snippet: Surface expression of Sbi domains D3D4. Serial dilutions of cells were applied to a nitrocellulose membrane and probed with rabbit anti-Sbi D3D4WrY IgG followed by HRP-conjugated goat anti-rabbit IgG.

    Article Snippet: Bound LTA was detected with an LTA (polyglycerolphosphate)-specific monoclonal antibody (clone 55, HyCult Biotechnology) followed by HRP-linked anti-mouse IgG (Cell Signalling).

    Techniques: Expressing

    Effects of hypoxia on StAR, P450scc and 3β-HSD protein by non-luteinizing and luteinizing granulosa cells. The cells were cultured under 20% O 2 or 10% O 2 (A and B) and 20% O 2 or 5% O 2 (C and D) for 24 h in the presence or absence of insulin (2 µg/ml), forskolin (10 µM) or insulin (2 µg/ml) in combination with forskolin (10 µM). Representative samples of Western blotting for StAR, P450scc, 3β-HSD and β-actin are shown in Fig. 5A for 10% O 2 and in Fig. 5C for 5% O 2 . The blot was incubated with primary antibodies against StAR, P450scc, 3β-HSD or β-actin and then incubated with secondary antibody conjugated to HRP. The resultant signal was detected by chemiluminescence and quantitated by computer-assisted densitometry. All protein levels are expressed relative to the amounts of β-actin. Asterisks indicate significant differences (P

    Journal: The Journal of Reproduction and Development

    Article Title: Hypoxia Promotes Progesterone Synthesis During Luteinization in Bovine Granulosa Cells

    doi: 10.1262/jrd.2014-014

    Figure Lengend Snippet: Effects of hypoxia on StAR, P450scc and 3β-HSD protein by non-luteinizing and luteinizing granulosa cells. The cells were cultured under 20% O 2 or 10% O 2 (A and B) and 20% O 2 or 5% O 2 (C and D) for 24 h in the presence or absence of insulin (2 µg/ml), forskolin (10 µM) or insulin (2 µg/ml) in combination with forskolin (10 µM). Representative samples of Western blotting for StAR, P450scc, 3β-HSD and β-actin are shown in Fig. 5A for 10% O 2 and in Fig. 5C for 5% O 2 . The blot was incubated with primary antibodies against StAR, P450scc, 3β-HSD or β-actin and then incubated with secondary antibody conjugated to HRP. The resultant signal was detected by chemiluminescence and quantitated by computer-assisted densitometry. All protein levels are expressed relative to the amounts of β-actin. Asterisks indicate significant differences (P

    Article Snippet: The membranes were then incubated separately with a primary antibody in immunoreaction enhancer solution (Toyobo, Osaka, Japan; NKB-101) specific to each protein, HIF-1α antibody (Sigma-Aldrich; SAB2104366; 1:500), StAR antibody (Abcam; ab96637; 1:3,000), P450scc antibody (Abcam; ab75497; 1:1,000), 3β-HSD antibody (Abcam; ab75710; 1:3,000) and β-actin antibody (ACTB; Sigma-Aldrich; A2228; 1:8,000), for overnight at 4 C. The membranes were washed three times for 5 min in TBS-T at room temperature, incubated with a secondary antibody in immunoreaction enhancer solution (for HIF-1α, StAR and P450scc [1:5,000], anti-rabbit Ig, HRP-linked whole antibody produced in donkey; Amersham Biosciences, Piscataway, NJ, USA; NA934; for 3β-HSD and ACTB [1:40,000], anti-mouse Ig, HRP-linked whole antibody produced in sheep; Amersham Biosciences; NA931) for 1 h and washed three times in TBS-T for 5 min at room temperature.

    Techniques: Cell Culture, Western Blot, Incubation