an2-cys Search Results


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  • 99
    ATCC desulfovibrio gigas
    Desulfovibrio Gigas, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti vinculin antibody
    Anti Vinculin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 665 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Chem Impex International fmocamino acids
    Fmocamino Acids, supplied by Chem Impex International, used in various techniques. Bioz Stars score: 78/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Millipore unnatural pyroglutamic
    Unnatural Pyroglutamic, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Proteintech symphony peptide synthesizer
    Symphony Peptide Synthesizer, supplied by Proteintech, used in various techniques. Bioz Stars score: 88/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Waters Corporation acquity uplc
    Acquity Uplc, supplied by Waters Corporation, used in various techniques. Bioz Stars score: 99/100, based on 1908 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Waters Corporation beh phenyl column
    Beh Phenyl Column, supplied by Waters Corporation, used in various techniques. Bioz Stars score: 97/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Abcam anti 2 cys prx antibodies
    Expression of CSE, and effect of Na 2 S on <t>2-Cys</t> <t>Prx</t> sulfinylation. A, Left, RAW264.7 cells were left untreated or stimulated for 16 h with 200 ng/mL LPS for 16 h. Cell lysates were run on SDS-PAGE and immunoblotted for CSE. (A) Right, RAW264.7 cells were left untreated or stimulated for 16 h with 200 ng/mL LPS for 16 h in the presence or absence of 1 mM PAG. After exhaustive washing, cells were challenged with 100 µM H 2 O 2 for 20 min. Cell lysates were run on SDS-PAGE and immunoblotted for 2-Cys Prx-SO 2/3 . (B) RAW264.7 cells were exposed to Na 2 S for 30 min and, after washing, to 100 µM H 2 O 2 for 20 min. Lysates were collected, and 2-Cys Prx-SO 2/3 expression was analyzed by immunoblotting using a specific antibody. Anti-β-actin antibody was used as a loading control. Results are representative of 3 independent experiments.
    Anti 2 Cys Prx Antibodies, supplied by Abcam, used in various techniques. Bioz Stars score: 80/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC d desulfuricans atcc 27774
    Expression of CSE, and effect of Na 2 S on <t>2-Cys</t> <t>Prx</t> sulfinylation. A, Left, RAW264.7 cells were left untreated or stimulated for 16 h with 200 ng/mL LPS for 16 h. Cell lysates were run on SDS-PAGE and immunoblotted for CSE. (A) Right, RAW264.7 cells were left untreated or stimulated for 16 h with 200 ng/mL LPS for 16 h in the presence or absence of 1 mM PAG. After exhaustive washing, cells were challenged with 100 µM H 2 O 2 for 20 min. Cell lysates were run on SDS-PAGE and immunoblotted for 2-Cys Prx-SO 2/3 . (B) RAW264.7 cells were exposed to Na 2 S for 30 min and, after washing, to 100 µM H 2 O 2 for 20 min. Lysates were collected, and 2-Cys Prx-SO 2/3 expression was analyzed by immunoblotting using a specific antibody. Anti-β-actin antibody was used as a loading control. Results are representative of 3 independent experiments.
    D Desulfuricans Atcc 27774, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 106 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti 2 cys peroxiredoxin
    Expression of CSE, and effect of Na 2 S on <t>2-Cys</t> <t>Prx</t> sulfinylation. A, Left, RAW264.7 cells were left untreated or stimulated for 16 h with 200 ng/mL LPS for 16 h. Cell lysates were run on SDS-PAGE and immunoblotted for CSE. (A) Right, RAW264.7 cells were left untreated or stimulated for 16 h with 200 ng/mL LPS for 16 h in the presence or absence of 1 mM PAG. After exhaustive washing, cells were challenged with 100 µM H 2 O 2 for 20 min. Cell lysates were run on SDS-PAGE and immunoblotted for 2-Cys Prx-SO 2/3 . (B) RAW264.7 cells were exposed to Na 2 S for 30 min and, after washing, to 100 µM H 2 O 2 for 20 min. Lysates were collected, and 2-Cys Prx-SO 2/3 expression was analyzed by immunoblotting using a specific antibody. Anti-β-actin antibody was used as a loading control. Results are representative of 3 independent experiments.
    Anti 2 Cys Peroxiredoxin, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa pmd 18t vector
    Expression of CSE, and effect of Na 2 S on <t>2-Cys</t> <t>Prx</t> sulfinylation. A, Left, RAW264.7 cells were left untreated or stimulated for 16 h with 200 ng/mL LPS for 16 h. Cell lysates were run on SDS-PAGE and immunoblotted for CSE. (A) Right, RAW264.7 cells were left untreated or stimulated for 16 h with 200 ng/mL LPS for 16 h in the presence or absence of 1 mM PAG. After exhaustive washing, cells were challenged with 100 µM H 2 O 2 for 20 min. Cell lysates were run on SDS-PAGE and immunoblotted for 2-Cys Prx-SO 2/3 . (B) RAW264.7 cells were exposed to Na 2 S for 30 min and, after washing, to 100 µM H 2 O 2 for 20 min. Lysates were collected, and 2-Cys Prx-SO 2/3 expression was analyzed by immunoblotting using a specific antibody. Anti-β-actin antibody was used as a loading control. Results are representative of 3 independent experiments.
    Pmd 18t Vector, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 1854 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega pgem t easy vector
    Expression of CSE, and effect of Na 2 S on <t>2-Cys</t> <t>Prx</t> sulfinylation. A, Left, RAW264.7 cells were left untreated or stimulated for 16 h with 200 ng/mL LPS for 16 h. Cell lysates were run on SDS-PAGE and immunoblotted for CSE. (A) Right, RAW264.7 cells were left untreated or stimulated for 16 h with 200 ng/mL LPS for 16 h in the presence or absence of 1 mM PAG. After exhaustive washing, cells were challenged with 100 µM H 2 O 2 for 20 min. Cell lysates were run on SDS-PAGE and immunoblotted for 2-Cys Prx-SO 2/3 . (B) RAW264.7 cells were exposed to Na 2 S for 30 min and, after washing, to 100 µM H 2 O 2 for 20 min. Lysates were collected, and 2-Cys Prx-SO 2/3 expression was analyzed by immunoblotting using a specific antibody. Anti-β-actin antibody was used as a loading control. Results are representative of 3 independent experiments.
    Pgem T Easy Vector, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 68436 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Abfrontier 2 cys prx antibodies
    Expression of CSE, and effect of Na 2 S on <t>2-Cys</t> <t>Prx</t> sulfinylation. A, Left, RAW264.7 cells were left untreated or stimulated for 16 h with 200 ng/mL LPS for 16 h. Cell lysates were run on SDS-PAGE and immunoblotted for CSE. (A) Right, RAW264.7 cells were left untreated or stimulated for 16 h with 200 ng/mL LPS for 16 h in the presence or absence of 1 mM PAG. After exhaustive washing, cells were challenged with 100 µM H 2 O 2 for 20 min. Cell lysates were run on SDS-PAGE and immunoblotted for 2-Cys Prx-SO 2/3 . (B) RAW264.7 cells were exposed to Na 2 S for 30 min and, after washing, to 100 µM H 2 O 2 for 20 min. Lysates were collected, and 2-Cys Prx-SO 2/3 expression was analyzed by immunoblotting using a specific antibody. Anti-β-actin antibody was used as a loading control. Results are representative of 3 independent experiments.
    2 Cys Prx Antibodies, supplied by Abfrontier, used in various techniques. Bioz Stars score: 80/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher maxima first strand cdna synthesis kit
    Expression of CSE, and effect of Na 2 S on <t>2-Cys</t> <t>Prx</t> sulfinylation. A, Left, RAW264.7 cells were left untreated or stimulated for 16 h with 200 ng/mL LPS for 16 h. Cell lysates were run on SDS-PAGE and immunoblotted for CSE. (A) Right, RAW264.7 cells were left untreated or stimulated for 16 h with 200 ng/mL LPS for 16 h in the presence or absence of 1 mM PAG. After exhaustive washing, cells were challenged with 100 µM H 2 O 2 for 20 min. Cell lysates were run on SDS-PAGE and immunoblotted for 2-Cys Prx-SO 2/3 . (B) RAW264.7 cells were exposed to Na 2 S for 30 min and, after washing, to 100 µM H 2 O 2 for 20 min. Lysates were collected, and 2-Cys Prx-SO 2/3 expression was analyzed by immunoblotting using a specific antibody. Anti-β-actin antibody was used as a loading control. Results are representative of 3 independent experiments.
    Maxima First Strand Cdna Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7759 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher supersignal west pico chemiluminescent substrate
    Expression of CSE, and effect of Na 2 S on <t>2-Cys</t> <t>Prx</t> sulfinylation. A, Left, RAW264.7 cells were left untreated or stimulated for 16 h with 200 ng/mL LPS for 16 h. Cell lysates were run on SDS-PAGE and immunoblotted for CSE. (A) Right, RAW264.7 cells were left untreated or stimulated for 16 h with 200 ng/mL LPS for 16 h in the presence or absence of 1 mM PAG. After exhaustive washing, cells were challenged with 100 µM H 2 O 2 for 20 min. Cell lysates were run on SDS-PAGE and immunoblotted for 2-Cys Prx-SO 2/3 . (B) RAW264.7 cells were exposed to Na 2 S for 30 min and, after washing, to 100 µM H 2 O 2 for 20 min. Lysates were collected, and 2-Cys Prx-SO 2/3 expression was analyzed by immunoblotting using a specific antibody. Anti-β-actin antibody was used as a loading control. Results are representative of 3 independent experiments.
    Supersignal West Pico Chemiluminescent Substrate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 30308 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore polyvinylidene difluoride membranes
    Expression of CSE, and effect of Na 2 S on <t>2-Cys</t> <t>Prx</t> sulfinylation. A, Left, RAW264.7 cells were left untreated or stimulated for 16 h with 200 ng/mL LPS for 16 h. Cell lysates were run on SDS-PAGE and immunoblotted for CSE. (A) Right, RAW264.7 cells were left untreated or stimulated for 16 h with 200 ng/mL LPS for 16 h in the presence or absence of 1 mM PAG. After exhaustive washing, cells were challenged with 100 µM H 2 O 2 for 20 min. Cell lysates were run on SDS-PAGE and immunoblotted for 2-Cys Prx-SO 2/3 . (B) RAW264.7 cells were exposed to Na 2 S for 30 min and, after washing, to 100 µM H 2 O 2 for 20 min. Lysates were collected, and 2-Cys Prx-SO 2/3 expression was analyzed by immunoblotting using a specific antibody. Anti-β-actin antibody was used as a loading control. Results are representative of 3 independent experiments.
    Polyvinylidene Difluoride Membranes, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 29842 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Qiagen pqe32 vector
    Expression of CSE, and effect of Na 2 S on <t>2-Cys</t> <t>Prx</t> sulfinylation. A, Left, RAW264.7 cells were left untreated or stimulated for 16 h with 200 ng/mL LPS for 16 h. Cell lysates were run on SDS-PAGE and immunoblotted for CSE. (A) Right, RAW264.7 cells were left untreated or stimulated for 16 h with 200 ng/mL LPS for 16 h in the presence or absence of 1 mM PAG. After exhaustive washing, cells were challenged with 100 µM H 2 O 2 for 20 min. Cell lysates were run on SDS-PAGE and immunoblotted for 2-Cys Prx-SO 2/3 . (B) RAW264.7 cells were exposed to Na 2 S for 30 min and, after washing, to 100 µM H 2 O 2 for 20 min. Lysates were collected, and 2-Cys Prx-SO 2/3 expression was analyzed by immunoblotting using a specific antibody. Anti-β-actin antibody was used as a loading control. Results are representative of 3 independent experiments.
    Pqe32 Vector, supplied by Qiagen, used in various techniques. Bioz Stars score: 95/100, based on 205 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher trizol reagent
    Expression of CSE, and effect of Na 2 S on <t>2-Cys</t> <t>Prx</t> sulfinylation. A, Left, RAW264.7 cells were left untreated or stimulated for 16 h with 200 ng/mL LPS for 16 h. Cell lysates were run on SDS-PAGE and immunoblotted for CSE. (A) Right, RAW264.7 cells were left untreated or stimulated for 16 h with 200 ng/mL LPS for 16 h in the presence or absence of 1 mM PAG. After exhaustive washing, cells were challenged with 100 µM H 2 O 2 for 20 min. Cell lysates were run on SDS-PAGE and immunoblotted for 2-Cys Prx-SO 2/3 . (B) RAW264.7 cells were exposed to Na 2 S for 30 min and, after washing, to 100 µM H 2 O 2 for 20 min. Lysates were collected, and 2-Cys Prx-SO 2/3 expression was analyzed by immunoblotting using a specific antibody. Anti-β-actin antibody was used as a loading control. Results are representative of 3 independent experiments.
    Trizol Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 465680 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Abcam anti 2 cys prx so2 3 antibodies
    Variation of Prx1 in RAW264.7 cells after treatment with H 2 O 2 or NO. (A–C) Cell extracts were separated by 2-D gel on 11-cm strip 7–10 NL pH gradients followed by immunodetection. Only the region of the gel containing Prx1 (basic pI) is shown. A, Extracts from cells exposed to DETA-NO or H 2 O 2 or to both added in sequence (as described in Fig. 2 ). Original (reduced) Prx1 and sulfinylated Prx1 spots were detected with an anti-Prx1 antibody (red) and an <t>anti-2-Cys</t> <t>Prx-SO</t> 2 / 3 antibody (green), respectively. (B) Lysates prepared from RAW264.7 cells exposed to 400 µM Cys-NO in HBBS for 30 min at 37 °C. The various forms of Prx1 were detected with an anti-Prx1 antibody. (C) Lysates prepared from RAW264.7 cells were stimulated with 20 U/mL IFN-γ and 200 ng/mL LPS or exposed to 500 µM DETA-NO for 16 h. The various forms of Prx1 were detected with an anti-Prx1 antibody. Original Prx1 spots were labeled with an arrow pointing up whereas acidic-shifted spots were labeled with an arrow pointing down.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
    Anti 2 Cys Prx So2 3 Antibodies, supplied by Abcam, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    TaKaRa pt7 blue vector
    Variation of Prx1 in RAW264.7 cells after treatment with H 2 O 2 or NO. (A–C) Cell extracts were separated by 2-D gel on 11-cm strip 7–10 NL pH gradients followed by immunodetection. Only the region of the gel containing Prx1 (basic pI) is shown. A, Extracts from cells exposed to DETA-NO or H 2 O 2 or to both added in sequence (as described in Fig. 2 ). Original (reduced) Prx1 and sulfinylated Prx1 spots were detected with an anti-Prx1 antibody (red) and an <t>anti-2-Cys</t> <t>Prx-SO</t> 2 / 3 antibody (green), respectively. (B) Lysates prepared from RAW264.7 cells exposed to 400 µM Cys-NO in HBBS for 30 min at 37 °C. The various forms of Prx1 were detected with an anti-Prx1 antibody. (C) Lysates prepared from RAW264.7 cells were stimulated with 20 U/mL IFN-γ and 200 ng/mL LPS or exposed to 500 µM DETA-NO for 16 h. The various forms of Prx1 were detected with an anti-Prx1 antibody. Original Prx1 spots were labeled with an arrow pointing up whereas acidic-shifted spots were labeled with an arrow pointing down.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
    Pt7 Blue Vector, supplied by TaKaRa, used in various techniques. Bioz Stars score: 84/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    prxs  (Abcam)
    79
    Abcam prxs
    Variation of Prx1 in RAW264.7 cells after treatment with H 2 O 2 or NO. (A–C) Cell extracts were separated by 2-D gel on 11-cm strip 7–10 NL pH gradients followed by immunodetection. Only the region of the gel containing Prx1 (basic pI) is shown. A, Extracts from cells exposed to DETA-NO or H 2 O 2 or to both added in sequence (as described in Fig. 2 ). Original (reduced) Prx1 and sulfinylated Prx1 spots were detected with an anti-Prx1 antibody (red) and an <t>anti-2-Cys</t> <t>Prx-SO</t> 2 / 3 antibody (green), respectively. (B) Lysates prepared from RAW264.7 cells exposed to 400 µM Cys-NO in HBBS for 30 min at 37 °C. The various forms of Prx1 were detected with an anti-Prx1 antibody. (C) Lysates prepared from RAW264.7 cells were stimulated with 20 U/mL IFN-γ and 200 ng/mL LPS or exposed to 500 µM DETA-NO for 16 h. The various forms of Prx1 were detected with an anti-Prx1 antibody. Original Prx1 spots were labeled with an arrow pointing up whereas acidic-shifted spots were labeled with an arrow pointing down.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
    Prxs, supplied by Abcam, used in various techniques. Bioz Stars score: 79/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore trx o c terminal peptide arlnhiteklfkkd
    Variation of Prx1 in RAW264.7 cells after treatment with H 2 O 2 or NO. (A–C) Cell extracts were separated by 2-D gel on 11-cm strip 7–10 NL pH gradients followed by immunodetection. Only the region of the gel containing Prx1 (basic pI) is shown. A, Extracts from cells exposed to DETA-NO or H 2 O 2 or to both added in sequence (as described in Fig. 2 ). Original (reduced) Prx1 and sulfinylated Prx1 spots were detected with an anti-Prx1 antibody (red) and an <t>anti-2-Cys</t> <t>Prx-SO</t> 2 / 3 antibody (green), respectively. (B) Lysates prepared from RAW264.7 cells exposed to 400 µM Cys-NO in HBBS for 30 min at 37 °C. The various forms of Prx1 were detected with an anti-Prx1 antibody. (C) Lysates prepared from RAW264.7 cells were stimulated with 20 U/mL IFN-γ and 200 ng/mL LPS or exposed to 500 µM DETA-NO for 16 h. The various forms of Prx1 were detected with an anti-Prx1 antibody. Original Prx1 spots were labeled with an arrow pointing up whereas acidic-shifted spots were labeled with an arrow pointing down.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
    Trx O C Terminal Peptide Arlnhiteklfkkd, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    AnaSpec cys 2 cys 7 disulfide bond
    Variation of Prx1 in RAW264.7 cells after treatment with H 2 O 2 or NO. (A–C) Cell extracts were separated by 2-D gel on 11-cm strip 7–10 NL pH gradients followed by immunodetection. Only the region of the gel containing Prx1 (basic pI) is shown. A, Extracts from cells exposed to DETA-NO or H 2 O 2 or to both added in sequence (as described in Fig. 2 ). Original (reduced) Prx1 and sulfinylated Prx1 spots were detected with an anti-Prx1 antibody (red) and an <t>anti-2-Cys</t> <t>Prx-SO</t> 2 / 3 antibody (green), respectively. (B) Lysates prepared from RAW264.7 cells exposed to 400 µM Cys-NO in HBBS for 30 min at 37 °C. The various forms of Prx1 were detected with an anti-Prx1 antibody. (C) Lysates prepared from RAW264.7 cells were stimulated with 20 U/mL IFN-γ and 200 ng/mL LPS or exposed to 500 µM DETA-NO for 16 h. The various forms of Prx1 were detected with an anti-Prx1 antibody. Original Prx1 spots were labeled with an arrow pointing up whereas acidic-shifted spots were labeled with an arrow pointing down.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
    Cys 2 Cys 7 Disulfide Bond, supplied by AnaSpec, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    GE Healthcare cyanine cy 2 cy 3 labeled antibodies
    Variation of Prx1 in RAW264.7 cells after treatment with H 2 O 2 or NO. (A–C) Cell extracts were separated by 2-D gel on 11-cm strip 7–10 NL pH gradients followed by immunodetection. Only the region of the gel containing Prx1 (basic pI) is shown. A, Extracts from cells exposed to DETA-NO or H 2 O 2 or to both added in sequence (as described in Fig. 2 ). Original (reduced) Prx1 and sulfinylated Prx1 spots were detected with an anti-Prx1 antibody (red) and an <t>anti-2-Cys</t> <t>Prx-SO</t> 2 / 3 antibody (green), respectively. (B) Lysates prepared from RAW264.7 cells exposed to 400 µM Cys-NO in HBBS for 30 min at 37 °C. The various forms of Prx1 were detected with an anti-Prx1 antibody. (C) Lysates prepared from RAW264.7 cells were stimulated with 20 U/mL IFN-γ and 200 ng/mL LPS or exposed to 500 µM DETA-NO for 16 h. The various forms of Prx1 were detected with an anti-Prx1 antibody. Original Prx1 spots were labeled with an arrow pointing up whereas acidic-shifted spots were labeled with an arrow pointing down.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
    Cyanine Cy 2 Cy 3 Labeled Antibodies, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 84/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher abi prism 7900 ht sequence detection system
    Variation of Prx1 in RAW264.7 cells after treatment with H 2 O 2 or NO. (A–C) Cell extracts were separated by 2-D gel on 11-cm strip 7–10 NL pH gradients followed by immunodetection. Only the region of the gel containing Prx1 (basic pI) is shown. A, Extracts from cells exposed to DETA-NO or H 2 O 2 or to both added in sequence (as described in Fig. 2 ). Original (reduced) Prx1 and sulfinylated Prx1 spots were detected with an anti-Prx1 antibody (red) and an <t>anti-2-Cys</t> <t>Prx-SO</t> 2 / 3 antibody (green), respectively. (B) Lysates prepared from RAW264.7 cells exposed to 400 µM Cys-NO in HBBS for 30 min at 37 °C. The various forms of Prx1 were detected with an anti-Prx1 antibody. (C) Lysates prepared from RAW264.7 cells were stimulated with 20 U/mL IFN-γ and 200 ng/mL LPS or exposed to 500 µM DETA-NO for 16 h. The various forms of Prx1 were detected with an anti-Prx1 antibody. Original Prx1 spots were labeled with an arrow pointing up whereas acidic-shifted spots were labeled with an arrow pointing down.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
    Abi Prism 7900 Ht Sequence Detection System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4883 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology b7 2
    Models for the interaction of CTLA-4 and CD28 with B7-1 and <t>B7-2.</t> ( A ) Steady-state distribution of B7-1 (dimer) and B7-2 (monomer). ( B ) Assemblies of B7-1 and B7-2 upon binding to CTLA-4. At low concentration of CTLA-4 (relative to B7-1), a bivalent homodimeric CTLA-4 molecule can bind two B7-1 molecules, but, at high concentration of CTLA-4, it may form an extended array with B7-1. However, with monomeric B7-2, CTLA-4 can engage only two B7-2 monomers. ( C ) Assemblies of B7-1 and B7-2 upon binding to CD28. At low concentration of monovalent CD28, a dimeric B7-1 could engage a single CD28 molecule; however, at high concentration of CD28, two molecules of CD28 may be bridged by one molecule of B7-1 without the possibility of forming higher order assemblies. With monomeric B7-2, monovalent CD28 may form single solitary complexes. ( D ) Assemblies of B7-1 and B7-2 upon cross-linking by bivalent monoclonal antibodies. Anti-B7-1 antibodies may interact with dimeric B7-1 like CTLA-4 and form an ordered network whereas anti-B7-2 antibodies may simply induce bridging of two B7-2 monomers. ( E ) Key for the symbols.
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    Abcam anti prx1
    S-homocysteinylation site identification in <t>Prx1,</t> by mass spectrometry. RAW264.7 cells were exposed to 500 µM DETA-NO for 16 h. Cell lysates were analyzed by 2-D SDS-PAGE gel followed by LC–MS/MS. Upper and bottom panels shows respectively MS/MS spectra of unmodified and modified Cys-52-containing peptide 38–62 from Prx1; mass spectra of the 2 triply charged peptides are shown in the right-hand corner, with monoisotopics m / z 1031.8424 (mass accuracy Δ m = 0.63 ppm) and 1057.1783 (mass accuracy Δ m = 3.61 ppm). The identified fragments are annotated on the sequences. Mass difference between y11 and y12 fragments indicates carbamidomethylation (Δ m = 57 Da) and S-homocysteinylation (Δ m = 133.01 Da) on cysteine for unmodified and modified peptide respectively. Mascot scores of 2 peptides (90 and 63) are significantly higher than the identity threshold [37] . Modified peptide by homocysteine was not detected in the gel spot after treatment with DTT and iodoacetamide. Shown are data representative of three independent experiments.
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    Thermo Fisher amplex red hydrogen peroxide peroxidase assay kit
    S-homocysteinylation site identification in <t>Prx1,</t> by mass spectrometry. RAW264.7 cells were exposed to 500 µM DETA-NO for 16 h. Cell lysates were analyzed by 2-D SDS-PAGE gel followed by LC–MS/MS. Upper and bottom panels shows respectively MS/MS spectra of unmodified and modified Cys-52-containing peptide 38–62 from Prx1; mass spectra of the 2 triply charged peptides are shown in the right-hand corner, with monoisotopics m / z 1031.8424 (mass accuracy Δ m = 0.63 ppm) and 1057.1783 (mass accuracy Δ m = 3.61 ppm). The identified fragments are annotated on the sequences. Mass difference between y11 and y12 fragments indicates carbamidomethylation (Δ m = 57 Da) and S-homocysteinylation (Δ m = 133.01 Da) on cysteine for unmodified and modified peptide respectively. Mascot scores of 2 peptides (90 and 63) are significantly higher than the identity threshold [37] . Modified peptide by homocysteine was not detected in the gel spot after treatment with DTT and iodoacetamide. Shown are data representative of three independent experiments.
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    Agrisera prk antibody
    S-homocysteinylation site identification in <t>Prx1,</t> by mass spectrometry. RAW264.7 cells were exposed to 500 µM DETA-NO for 16 h. Cell lysates were analyzed by 2-D SDS-PAGE gel followed by LC–MS/MS. Upper and bottom panels shows respectively MS/MS spectra of unmodified and modified Cys-52-containing peptide 38–62 from Prx1; mass spectra of the 2 triply charged peptides are shown in the right-hand corner, with monoisotopics m / z 1031.8424 (mass accuracy Δ m = 0.63 ppm) and 1057.1783 (mass accuracy Δ m = 3.61 ppm). The identified fragments are annotated on the sequences. Mass difference between y11 and y12 fragments indicates carbamidomethylation (Δ m = 57 Da) and S-homocysteinylation (Δ m = 133.01 Da) on cysteine for unmodified and modified peptide respectively. Mascot scores of 2 peptides (90 and 63) are significantly higher than the identity threshold [37] . Modified peptide by homocysteine was not detected in the gel spot after treatment with DTT and iodoacetamide. Shown are data representative of three independent experiments.
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    Image Search Results


    Expression of CSE, and effect of Na 2 S on 2-Cys Prx sulfinylation. A, Left, RAW264.7 cells were left untreated or stimulated for 16 h with 200 ng/mL LPS for 16 h. Cell lysates were run on SDS-PAGE and immunoblotted for CSE. (A) Right, RAW264.7 cells were left untreated or stimulated for 16 h with 200 ng/mL LPS for 16 h in the presence or absence of 1 mM PAG. After exhaustive washing, cells were challenged with 100 µM H 2 O 2 for 20 min. Cell lysates were run on SDS-PAGE and immunoblotted for 2-Cys Prx-SO 2/3 . (B) RAW264.7 cells were exposed to Na 2 S for 30 min and, after washing, to 100 µM H 2 O 2 for 20 min. Lysates were collected, and 2-Cys Prx-SO 2/3 expression was analyzed by immunoblotting using a specific antibody. Anti-β-actin antibody was used as a loading control. Results are representative of 3 independent experiments.

    Journal: Redox Biology

    Article Title: Peroxiredoxin post-translational modifications by redox messengers

    doi: 10.1016/j.redox.2014.06.001

    Figure Lengend Snippet: Expression of CSE, and effect of Na 2 S on 2-Cys Prx sulfinylation. A, Left, RAW264.7 cells were left untreated or stimulated for 16 h with 200 ng/mL LPS for 16 h. Cell lysates were run on SDS-PAGE and immunoblotted for CSE. (A) Right, RAW264.7 cells were left untreated or stimulated for 16 h with 200 ng/mL LPS for 16 h in the presence or absence of 1 mM PAG. After exhaustive washing, cells were challenged with 100 µM H 2 O 2 for 20 min. Cell lysates were run on SDS-PAGE and immunoblotted for 2-Cys Prx-SO 2/3 . (B) RAW264.7 cells were exposed to Na 2 S for 30 min and, after washing, to 100 µM H 2 O 2 for 20 min. Lysates were collected, and 2-Cys Prx-SO 2/3 expression was analyzed by immunoblotting using a specific antibody. Anti-β-actin antibody was used as a loading control. Results are representative of 3 independent experiments.

    Article Snippet: After electrophoresis and protein immobilization, polyvinylidene difluoride membranes (Millipore) were blocked with nonfat milk and incubated with primary antibodies: anti-CSE (CTH 30.7) monoclonal antibody from Santa Cruz, anti-β-actin from Sigma-Aldrich, and anti-2-Cys Prx-SO2/3 and anti-2-Cys Prx antibodies from Abcam.

    Techniques: Expressing, SDS Page

    Variation of Prx1 in RAW264.7 cells after treatment with H 2 O 2 or NO. (A–C) Cell extracts were separated by 2-D gel on 11-cm strip 7–10 NL pH gradients followed by immunodetection. Only the region of the gel containing Prx1 (basic pI) is shown. A, Extracts from cells exposed to DETA-NO or H 2 O 2 or to both added in sequence (as described in Fig. 2 ). Original (reduced) Prx1 and sulfinylated Prx1 spots were detected with an anti-Prx1 antibody (red) and an anti-2-Cys Prx-SO 2 / 3 antibody (green), respectively. (B) Lysates prepared from RAW264.7 cells exposed to 400 µM Cys-NO in HBBS for 30 min at 37 °C. The various forms of Prx1 were detected with an anti-Prx1 antibody. (C) Lysates prepared from RAW264.7 cells were stimulated with 20 U/mL IFN-γ and 200 ng/mL LPS or exposed to 500 µM DETA-NO for 16 h. The various forms of Prx1 were detected with an anti-Prx1 antibody. Original Prx1 spots were labeled with an arrow pointing up whereas acidic-shifted spots were labeled with an arrow pointing down.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

    Journal: Redox Biology

    Article Title: Peroxiredoxin post-translational modifications by redox messengers

    doi: 10.1016/j.redox.2014.06.001

    Figure Lengend Snippet: Variation of Prx1 in RAW264.7 cells after treatment with H 2 O 2 or NO. (A–C) Cell extracts were separated by 2-D gel on 11-cm strip 7–10 NL pH gradients followed by immunodetection. Only the region of the gel containing Prx1 (basic pI) is shown. A, Extracts from cells exposed to DETA-NO or H 2 O 2 or to both added in sequence (as described in Fig. 2 ). Original (reduced) Prx1 and sulfinylated Prx1 spots were detected with an anti-Prx1 antibody (red) and an anti-2-Cys Prx-SO 2 / 3 antibody (green), respectively. (B) Lysates prepared from RAW264.7 cells exposed to 400 µM Cys-NO in HBBS for 30 min at 37 °C. The various forms of Prx1 were detected with an anti-Prx1 antibody. (C) Lysates prepared from RAW264.7 cells were stimulated with 20 U/mL IFN-γ and 200 ng/mL LPS or exposed to 500 µM DETA-NO for 16 h. The various forms of Prx1 were detected with an anti-Prx1 antibody. Original Prx1 spots were labeled with an arrow pointing up whereas acidic-shifted spots were labeled with an arrow pointing down.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

    Article Snippet: After electrophoresis and protein immobilization, polyvinylidene difluoride membranes (Millipore) were blocked with nonfat milk and incubated with primary antibodies: anti-CSE (CTH 30.7) monoclonal antibody from Santa Cruz, anti-β-actin from Sigma-Aldrich, and anti-2-Cys Prx-SO2/3 and anti-2-Cys Prx antibodies from Abcam.

    Techniques: Stripping Membranes, Immunodetection, Sequencing, Labeling

    Two-dimensional gel electrophoresis coupled to mass spectrometry or immunodetection for Prx identification. (A) Proteins (500 µg) extracted from RAW264.7 cells were separated by two-dimensional electrophoresis on 17-cm strip 3–10 NL pH gradients. Strips were loaded on 12% SDS-PAGE. The gel was stained with colloidal Coomassie blue, and 2-Cys Prx spots (indicated by arrows) were identified by MALDI-TOF. (B and C) Region of the 2-D gels containing the 2-Cys Prxs. Cell extracts (600 µg of protein) were prepared from RAW264.7 cells, either untreated (control) or exposed to 100 µM H 2 O 2 for 20 min with or without a 16-h pre-exposure to 500 µM DETA-NO. (B) Cell lysates were analyzed by 2-D SDS-PAGE gel followed by MALDI mass spectrometry. Spots corresponding to Prx 1 (basic pI) and Prx2 (acidic pI) are circled in red and marked with an arrow. Prx3 and Prx4 were not identified. (C) Cell extracts were analyzed by 2-D SDS-PAGE and 2-color immunodetection using an anti-Prx-SO 2/3 antibody (green color), and anti-2-Cys Prx and anti-Prx1 antibodies (red color). Additionally, anti-Prx1 antibody (red color) was also incubated with the membrane to maximize Prx1 signal. The yellowish-orange spots show an overlay of the reduced (red) and overoxidized (green) 2-Cys Prxs. White asterisks indicate extraneous spot artifacts.

    Journal: Redox Biology

    Article Title: Peroxiredoxin post-translational modifications by redox messengers

    doi: 10.1016/j.redox.2014.06.001

    Figure Lengend Snippet: Two-dimensional gel electrophoresis coupled to mass spectrometry or immunodetection for Prx identification. (A) Proteins (500 µg) extracted from RAW264.7 cells were separated by two-dimensional electrophoresis on 17-cm strip 3–10 NL pH gradients. Strips were loaded on 12% SDS-PAGE. The gel was stained with colloidal Coomassie blue, and 2-Cys Prx spots (indicated by arrows) were identified by MALDI-TOF. (B and C) Region of the 2-D gels containing the 2-Cys Prxs. Cell extracts (600 µg of protein) were prepared from RAW264.7 cells, either untreated (control) or exposed to 100 µM H 2 O 2 for 20 min with or without a 16-h pre-exposure to 500 µM DETA-NO. (B) Cell lysates were analyzed by 2-D SDS-PAGE gel followed by MALDI mass spectrometry. Spots corresponding to Prx 1 (basic pI) and Prx2 (acidic pI) are circled in red and marked with an arrow. Prx3 and Prx4 were not identified. (C) Cell extracts were analyzed by 2-D SDS-PAGE and 2-color immunodetection using an anti-Prx-SO 2/3 antibody (green color), and anti-2-Cys Prx and anti-Prx1 antibodies (red color). Additionally, anti-Prx1 antibody (red color) was also incubated with the membrane to maximize Prx1 signal. The yellowish-orange spots show an overlay of the reduced (red) and overoxidized (green) 2-Cys Prxs. White asterisks indicate extraneous spot artifacts.

    Article Snippet: After electrophoresis and protein immobilization, polyvinylidene difluoride membranes (Millipore) were blocked with nonfat milk and incubated with primary antibodies: anti-CSE (CTH 30.7) monoclonal antibody from Santa Cruz, anti-β-actin from Sigma-Aldrich, and anti-2-Cys Prx-SO2/3 and anti-2-Cys Prx antibodies from Abcam.

    Techniques: Two-Dimensional Gel Electrophoresis, Electrophoresis, Mass Spectrometry, Immunodetection, Stripping Membranes, SDS Page, Staining, Incubation

    Inhibition of HDAC activity does not preclude abatement of NO-dependent sulfinylation of 2-Cys Prxs. RAW264.7 cells were cultured in the presence of trichostatin A (TSA) for 16 h to inhibit HDAC activity or 500 µM DETA-NO after washings, cells were challenged with 100 µM H 2 O 2 for 20 min. Cell lysates were analyzed for expression of 2-Cys Prx-SO 2 by immunoblotting.

    Journal: Redox Biology

    Article Title: Peroxiredoxin post-translational modifications by redox messengers

    doi: 10.1016/j.redox.2014.06.001

    Figure Lengend Snippet: Inhibition of HDAC activity does not preclude abatement of NO-dependent sulfinylation of 2-Cys Prxs. RAW264.7 cells were cultured in the presence of trichostatin A (TSA) for 16 h to inhibit HDAC activity or 500 µM DETA-NO after washings, cells were challenged with 100 µM H 2 O 2 for 20 min. Cell lysates were analyzed for expression of 2-Cys Prx-SO 2 by immunoblotting.

    Article Snippet: After electrophoresis and protein immobilization, polyvinylidene difluoride membranes (Millipore) were blocked with nonfat milk and incubated with primary antibodies: anti-CSE (CTH 30.7) monoclonal antibody from Santa Cruz, anti-β-actin from Sigma-Aldrich, and anti-2-Cys Prx-SO2/3 and anti-2-Cys Prx antibodies from Abcam.

    Techniques: Inhibition, Activity Assay, Cell Culture, Expressing

    Variation of Prx1 in RAW264.7 cells after treatment with H 2 O 2 or NO. (A–C) Cell extracts were separated by 2-D gel on 11-cm strip 7–10 NL pH gradients followed by immunodetection. Only the region of the gel containing Prx1 (basic pI) is shown. A, Extracts from cells exposed to DETA-NO or H 2 O 2 or to both added in sequence (as described in Fig. 2 ). Original (reduced) Prx1 and sulfinylated Prx1 spots were detected with an anti-Prx1 antibody (red) and an anti-2-Cys Prx-SO 2 / 3 antibody (green), respectively. (B) Lysates prepared from RAW264.7 cells exposed to 400 µM Cys-NO in HBBS for 30 min at 37 °C. The various forms of Prx1 were detected with an anti-Prx1 antibody. (C) Lysates prepared from RAW264.7 cells were stimulated with 20 U/mL IFN-γ and 200 ng/mL LPS or exposed to 500 µM DETA-NO for 16 h. The various forms of Prx1 were detected with an anti-Prx1 antibody. Original Prx1 spots were labeled with an arrow pointing up whereas acidic-shifted spots were labeled with an arrow pointing down.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

    Journal: Redox Biology

    Article Title: Peroxiredoxin post-translational modifications by redox messengers

    doi: 10.1016/j.redox.2014.06.001

    Figure Lengend Snippet: Variation of Prx1 in RAW264.7 cells after treatment with H 2 O 2 or NO. (A–C) Cell extracts were separated by 2-D gel on 11-cm strip 7–10 NL pH gradients followed by immunodetection. Only the region of the gel containing Prx1 (basic pI) is shown. A, Extracts from cells exposed to DETA-NO or H 2 O 2 or to both added in sequence (as described in Fig. 2 ). Original (reduced) Prx1 and sulfinylated Prx1 spots were detected with an anti-Prx1 antibody (red) and an anti-2-Cys Prx-SO 2 / 3 antibody (green), respectively. (B) Lysates prepared from RAW264.7 cells exposed to 400 µM Cys-NO in HBBS for 30 min at 37 °C. The various forms of Prx1 were detected with an anti-Prx1 antibody. (C) Lysates prepared from RAW264.7 cells were stimulated with 20 U/mL IFN-γ and 200 ng/mL LPS or exposed to 500 µM DETA-NO for 16 h. The various forms of Prx1 were detected with an anti-Prx1 antibody. Original Prx1 spots were labeled with an arrow pointing up whereas acidic-shifted spots were labeled with an arrow pointing down.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

    Article Snippet: Antibodies and immunoblot analyses Anti-2-Cys Prx, anti-Prx1 and anti-2-Cys Prx-SO2/3 antibodies were from Abcam, and anti-vinculin antibody was from Sigma-Aldrich.

    Techniques: Stripping Membranes, Immunodetection, Sequencing, Labeling

    Two-dimensional gel electrophoresis coupled to mass spectrometry or immunodetection for Prx identification. (A) Proteins (500 µg) extracted from RAW264.7 cells were separated by two-dimensional electrophoresis on 17-cm strip 3–10 NL pH gradients. Strips were loaded on 12% SDS-PAGE. The gel was stained with colloidal Coomassie blue, and 2-Cys Prx spots (indicated by arrows) were identified by MALDI-TOF. (B and C) Region of the 2-D gels containing the 2-Cys Prxs. Cell extracts (600 µg of protein) were prepared from RAW264.7 cells, either untreated (control) or exposed to 100 µM H 2 O 2 for 20 min with or without a 16-h pre-exposure to 500 µM DETA-NO. (B) Cell lysates were analyzed by 2-D SDS-PAGE gel followed by MALDI mass spectrometry. Spots corresponding to Prx 1 (basic pI) and Prx2 (acidic pI) are circled in red and marked with an arrow. Prx3 and Prx4 were not identified. (C) Cell extracts were analyzed by 2-D SDS-PAGE and 2-color immunodetection using an anti-Prx-SO 2/3 antibody (green color), and anti-2-Cys Prx and anti-Prx1 antibodies (red color). Additionally, anti-Prx1 antibody (red color) was also incubated with the membrane to maximize Prx1 signal. The yellowish-orange spots show an overlay of the reduced (red) and overoxidized (green) 2-Cys Prxs. White asterisks indicate extraneous spot artifacts.

    Journal: Redox Biology

    Article Title: Peroxiredoxin post-translational modifications by redox messengers

    doi: 10.1016/j.redox.2014.06.001

    Figure Lengend Snippet: Two-dimensional gel electrophoresis coupled to mass spectrometry or immunodetection for Prx identification. (A) Proteins (500 µg) extracted from RAW264.7 cells were separated by two-dimensional electrophoresis on 17-cm strip 3–10 NL pH gradients. Strips were loaded on 12% SDS-PAGE. The gel was stained with colloidal Coomassie blue, and 2-Cys Prx spots (indicated by arrows) were identified by MALDI-TOF. (B and C) Region of the 2-D gels containing the 2-Cys Prxs. Cell extracts (600 µg of protein) were prepared from RAW264.7 cells, either untreated (control) or exposed to 100 µM H 2 O 2 for 20 min with or without a 16-h pre-exposure to 500 µM DETA-NO. (B) Cell lysates were analyzed by 2-D SDS-PAGE gel followed by MALDI mass spectrometry. Spots corresponding to Prx 1 (basic pI) and Prx2 (acidic pI) are circled in red and marked with an arrow. Prx3 and Prx4 were not identified. (C) Cell extracts were analyzed by 2-D SDS-PAGE and 2-color immunodetection using an anti-Prx-SO 2/3 antibody (green color), and anti-2-Cys Prx and anti-Prx1 antibodies (red color). Additionally, anti-Prx1 antibody (red color) was also incubated with the membrane to maximize Prx1 signal. The yellowish-orange spots show an overlay of the reduced (red) and overoxidized (green) 2-Cys Prxs. White asterisks indicate extraneous spot artifacts.

    Article Snippet: Antibodies and immunoblot analyses Anti-2-Cys Prx, anti-Prx1 and anti-2-Cys Prx-SO2/3 antibodies were from Abcam, and anti-vinculin antibody was from Sigma-Aldrich.

    Techniques: Two-Dimensional Gel Electrophoresis, Electrophoresis, Mass Spectrometry, Immunodetection, Stripping Membranes, SDS Page, Staining, Incubation

    Models for the interaction of CTLA-4 and CD28 with B7-1 and B7-2. ( A ) Steady-state distribution of B7-1 (dimer) and B7-2 (monomer). ( B ) Assemblies of B7-1 and B7-2 upon binding to CTLA-4. At low concentration of CTLA-4 (relative to B7-1), a bivalent homodimeric CTLA-4 molecule can bind two B7-1 molecules, but, at high concentration of CTLA-4, it may form an extended array with B7-1. However, with monomeric B7-2, CTLA-4 can engage only two B7-2 monomers. ( C ) Assemblies of B7-1 and B7-2 upon binding to CD28. At low concentration of monovalent CD28, a dimeric B7-1 could engage a single CD28 molecule; however, at high concentration of CD28, two molecules of CD28 may be bridged by one molecule of B7-1 without the possibility of forming higher order assemblies. With monomeric B7-2, monovalent CD28 may form single solitary complexes. ( D ) Assemblies of B7-1 and B7-2 upon cross-linking by bivalent monoclonal antibodies. Anti-B7-1 antibodies may interact with dimeric B7-1 like CTLA-4 and form an ordered network whereas anti-B7-2 antibodies may simply induce bridging of two B7-2 monomers. ( E ) Key for the symbols.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Different cell surface oligomeric states of B7-1 and B7-2: Implications for signaling

    doi: 10.1073/pnas.0507257102

    Figure Lengend Snippet: Models for the interaction of CTLA-4 and CD28 with B7-1 and B7-2. ( A ) Steady-state distribution of B7-1 (dimer) and B7-2 (monomer). ( B ) Assemblies of B7-1 and B7-2 upon binding to CTLA-4. At low concentration of CTLA-4 (relative to B7-1), a bivalent homodimeric CTLA-4 molecule can bind two B7-1 molecules, but, at high concentration of CTLA-4, it may form an extended array with B7-1. However, with monomeric B7-2, CTLA-4 can engage only two B7-2 monomers. ( C ) Assemblies of B7-1 and B7-2 upon binding to CD28. At low concentration of monovalent CD28, a dimeric B7-1 could engage a single CD28 molecule; however, at high concentration of CD28, two molecules of CD28 may be bridged by one molecule of B7-1 without the possibility of forming higher order assemblies. With monomeric B7-2, monovalent CD28 may form single solitary complexes. ( D ) Assemblies of B7-1 and B7-2 upon cross-linking by bivalent monoclonal antibodies. Anti-B7-1 antibodies may interact with dimeric B7-1 like CTLA-4 and form an ordered network whereas anti-B7-2 antibodies may simply induce bridging of two B7-2 monomers. ( E ) Key for the symbols.

    Article Snippet: By using a similar strategy, a cysteine trap mutant of B7-2 (B7-2:CYS) was generated with the rationale of trapping any population of dimers that might exist and that would be below the FRET detection limit.

    Techniques: Binding Assay, Concentration Assay

    S-homocysteinylation site identification in Prx1, by mass spectrometry. RAW264.7 cells were exposed to 500 µM DETA-NO for 16 h. Cell lysates were analyzed by 2-D SDS-PAGE gel followed by LC–MS/MS. Upper and bottom panels shows respectively MS/MS spectra of unmodified and modified Cys-52-containing peptide 38–62 from Prx1; mass spectra of the 2 triply charged peptides are shown in the right-hand corner, with monoisotopics m / z 1031.8424 (mass accuracy Δ m = 0.63 ppm) and 1057.1783 (mass accuracy Δ m = 3.61 ppm). The identified fragments are annotated on the sequences. Mass difference between y11 and y12 fragments indicates carbamidomethylation (Δ m = 57 Da) and S-homocysteinylation (Δ m = 133.01 Da) on cysteine for unmodified and modified peptide respectively. Mascot scores of 2 peptides (90 and 63) are significantly higher than the identity threshold [37] . Modified peptide by homocysteine was not detected in the gel spot after treatment with DTT and iodoacetamide. Shown are data representative of three independent experiments.

    Journal: Redox Biology

    Article Title: Peroxiredoxin post-translational modifications by redox messengers

    doi: 10.1016/j.redox.2014.06.001

    Figure Lengend Snippet: S-homocysteinylation site identification in Prx1, by mass spectrometry. RAW264.7 cells were exposed to 500 µM DETA-NO for 16 h. Cell lysates were analyzed by 2-D SDS-PAGE gel followed by LC–MS/MS. Upper and bottom panels shows respectively MS/MS spectra of unmodified and modified Cys-52-containing peptide 38–62 from Prx1; mass spectra of the 2 triply charged peptides are shown in the right-hand corner, with monoisotopics m / z 1031.8424 (mass accuracy Δ m = 0.63 ppm) and 1057.1783 (mass accuracy Δ m = 3.61 ppm). The identified fragments are annotated on the sequences. Mass difference between y11 and y12 fragments indicates carbamidomethylation (Δ m = 57 Da) and S-homocysteinylation (Δ m = 133.01 Da) on cysteine for unmodified and modified peptide respectively. Mascot scores of 2 peptides (90 and 63) are significantly higher than the identity threshold [37] . Modified peptide by homocysteine was not detected in the gel spot after treatment with DTT and iodoacetamide. Shown are data representative of three independent experiments.

    Article Snippet: Antibodies and immunoblot analyses Anti-2-Cys Prx, anti-Prx1 and anti-2-Cys Prx-SO2/3 antibodies were from Abcam, and anti-vinculin antibody was from Sigma-Aldrich.

    Techniques: Mass Spectrometry, SDS Page, Liquid Chromatography with Mass Spectroscopy, Modification

    The pattern of different isoelectric shifts of Prx1 in cells exposed to NO. RAW264.7 cells were exposed to 400 µM Cys-NO for 30 min in HBSS (A), or cultured for 16 h with 500 µM DETA-NO (B) or with a combination of 20 U/mL IFN-γ and 200 ng/mL LPS (C). When indicated, cell lysates were treated with 5 mM ascorbate and 10 µM CuSO 4 for 40 min at 37 °C. Cell lysates were then analyzed by 2-D SDS-PAGE and by immunodetection using an anti-Prx1 antibody and secondary antibodies coupled to IRDye 700. Images were collected and analyzed by using the LI-COR Odyssey infrared imaging system (Biosciences, Lincoln, NE, USA). Spots sensitive to ascorbate/copper are circled and marked by an open arrowhead pointing up (A and C), while the ascorbate/copper-resistant spot was referred to as X. Streaking toward the acidic extremity of the gel, where Prx2 is positioned, precluded proper interpretation.

    Journal: Redox Biology

    Article Title: Peroxiredoxin post-translational modifications by redox messengers

    doi: 10.1016/j.redox.2014.06.001

    Figure Lengend Snippet: The pattern of different isoelectric shifts of Prx1 in cells exposed to NO. RAW264.7 cells were exposed to 400 µM Cys-NO for 30 min in HBSS (A), or cultured for 16 h with 500 µM DETA-NO (B) or with a combination of 20 U/mL IFN-γ and 200 ng/mL LPS (C). When indicated, cell lysates were treated with 5 mM ascorbate and 10 µM CuSO 4 for 40 min at 37 °C. Cell lysates were then analyzed by 2-D SDS-PAGE and by immunodetection using an anti-Prx1 antibody and secondary antibodies coupled to IRDye 700. Images were collected and analyzed by using the LI-COR Odyssey infrared imaging system (Biosciences, Lincoln, NE, USA). Spots sensitive to ascorbate/copper are circled and marked by an open arrowhead pointing up (A and C), while the ascorbate/copper-resistant spot was referred to as X. Streaking toward the acidic extremity of the gel, where Prx2 is positioned, precluded proper interpretation.

    Article Snippet: Antibodies and immunoblot analyses Anti-2-Cys Prx, anti-Prx1 and anti-2-Cys Prx-SO2/3 antibodies were from Abcam, and anti-vinculin antibody was from Sigma-Aldrich.

    Techniques: Cell Culture, SDS Page, Immunodetection, Imaging

    Variation of Prx1 in RAW264.7 cells after treatment with H 2 O 2 or NO. (A–C) Cell extracts were separated by 2-D gel on 11-cm strip 7–10 NL pH gradients followed by immunodetection. Only the region of the gel containing Prx1 (basic pI) is shown. A, Extracts from cells exposed to DETA-NO or H 2 O 2 or to both added in sequence (as described in Fig. 2 ). Original (reduced) Prx1 and sulfinylated Prx1 spots were detected with an anti-Prx1 antibody (red) and an anti-2-Cys Prx-SO 2 / 3 antibody (green), respectively. (B) Lysates prepared from RAW264.7 cells exposed to 400 µM Cys-NO in HBBS for 30 min at 37 °C. The various forms of Prx1 were detected with an anti-Prx1 antibody. (C) Lysates prepared from RAW264.7 cells were stimulated with 20 U/mL IFN-γ and 200 ng/mL LPS or exposed to 500 µM DETA-NO for 16 h. The various forms of Prx1 were detected with an anti-Prx1 antibody. Original Prx1 spots were labeled with an arrow pointing up whereas acidic-shifted spots were labeled with an arrow pointing down.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

    Journal: Redox Biology

    Article Title: Peroxiredoxin post-translational modifications by redox messengers

    doi: 10.1016/j.redox.2014.06.001

    Figure Lengend Snippet: Variation of Prx1 in RAW264.7 cells after treatment with H 2 O 2 or NO. (A–C) Cell extracts were separated by 2-D gel on 11-cm strip 7–10 NL pH gradients followed by immunodetection. Only the region of the gel containing Prx1 (basic pI) is shown. A, Extracts from cells exposed to DETA-NO or H 2 O 2 or to both added in sequence (as described in Fig. 2 ). Original (reduced) Prx1 and sulfinylated Prx1 spots were detected with an anti-Prx1 antibody (red) and an anti-2-Cys Prx-SO 2 / 3 antibody (green), respectively. (B) Lysates prepared from RAW264.7 cells exposed to 400 µM Cys-NO in HBBS for 30 min at 37 °C. The various forms of Prx1 were detected with an anti-Prx1 antibody. (C) Lysates prepared from RAW264.7 cells were stimulated with 20 U/mL IFN-γ and 200 ng/mL LPS or exposed to 500 µM DETA-NO for 16 h. The various forms of Prx1 were detected with an anti-Prx1 antibody. Original Prx1 spots were labeled with an arrow pointing up whereas acidic-shifted spots were labeled with an arrow pointing down.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

    Article Snippet: Antibodies and immunoblot analyses Anti-2-Cys Prx, anti-Prx1 and anti-2-Cys Prx-SO2/3 antibodies were from Abcam, and anti-vinculin antibody was from Sigma-Aldrich.

    Techniques: Stripping Membranes, Immunodetection, Sequencing, Labeling

    Two-dimensional gel electrophoresis coupled to mass spectrometry or immunodetection for Prx identification. (A) Proteins (500 µg) extracted from RAW264.7 cells were separated by two-dimensional electrophoresis on 17-cm strip 3–10 NL pH gradients. Strips were loaded on 12% SDS-PAGE. The gel was stained with colloidal Coomassie blue, and 2-Cys Prx spots (indicated by arrows) were identified by MALDI-TOF. (B and C) Region of the 2-D gels containing the 2-Cys Prxs. Cell extracts (600 µg of protein) were prepared from RAW264.7 cells, either untreated (control) or exposed to 100 µM H 2 O 2 for 20 min with or without a 16-h pre-exposure to 500 µM DETA-NO. (B) Cell lysates were analyzed by 2-D SDS-PAGE gel followed by MALDI mass spectrometry. Spots corresponding to Prx 1 (basic pI) and Prx2 (acidic pI) are circled in red and marked with an arrow. Prx3 and Prx4 were not identified. (C) Cell extracts were analyzed by 2-D SDS-PAGE and 2-color immunodetection using an anti-Prx-SO 2/3 antibody (green color), and anti-2-Cys Prx and anti-Prx1 antibodies (red color). Additionally, anti-Prx1 antibody (red color) was also incubated with the membrane to maximize Prx1 signal. The yellowish-orange spots show an overlay of the reduced (red) and overoxidized (green) 2-Cys Prxs. White asterisks indicate extraneous spot artifacts.

    Journal: Redox Biology

    Article Title: Peroxiredoxin post-translational modifications by redox messengers

    doi: 10.1016/j.redox.2014.06.001

    Figure Lengend Snippet: Two-dimensional gel electrophoresis coupled to mass spectrometry or immunodetection for Prx identification. (A) Proteins (500 µg) extracted from RAW264.7 cells were separated by two-dimensional electrophoresis on 17-cm strip 3–10 NL pH gradients. Strips were loaded on 12% SDS-PAGE. The gel was stained with colloidal Coomassie blue, and 2-Cys Prx spots (indicated by arrows) were identified by MALDI-TOF. (B and C) Region of the 2-D gels containing the 2-Cys Prxs. Cell extracts (600 µg of protein) were prepared from RAW264.7 cells, either untreated (control) or exposed to 100 µM H 2 O 2 for 20 min with or without a 16-h pre-exposure to 500 µM DETA-NO. (B) Cell lysates were analyzed by 2-D SDS-PAGE gel followed by MALDI mass spectrometry. Spots corresponding to Prx 1 (basic pI) and Prx2 (acidic pI) are circled in red and marked with an arrow. Prx3 and Prx4 were not identified. (C) Cell extracts were analyzed by 2-D SDS-PAGE and 2-color immunodetection using an anti-Prx-SO 2/3 antibody (green color), and anti-2-Cys Prx and anti-Prx1 antibodies (red color). Additionally, anti-Prx1 antibody (red color) was also incubated with the membrane to maximize Prx1 signal. The yellowish-orange spots show an overlay of the reduced (red) and overoxidized (green) 2-Cys Prxs. White asterisks indicate extraneous spot artifacts.

    Article Snippet: Antibodies and immunoblot analyses Anti-2-Cys Prx, anti-Prx1 and anti-2-Cys Prx-SO2/3 antibodies were from Abcam, and anti-vinculin antibody was from Sigma-Aldrich.

    Techniques: Two-Dimensional Gel Electrophoresis, Electrophoresis, Mass Spectrometry, Immunodetection, Stripping Membranes, SDS Page, Staining, Incubation