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  • 92
    Santa Cruz Biotechnology hrp conjugated secondary antibodies
    siRNA directed toward the <t>uPA</t> promoter prevents radiation-induced hypomethylation. (A) Schematic depiction of the uPA promoter showing shRNA target region, Sp1 binding sites, and the region selected for MSP. (B) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, bisulfite-treated genomic DNA was subjected to MSP and followed by agarose gel analysis. (C) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, conditioned media were subjected to fibrin zymography, total RNA was used to perform RT-PCR, and cell lysates were used for immunoblot analysis as indicated. (D) The band intensities were quantified and represented as arbitrary units. (E) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, cells were fixed and subjected to immunocytochemistry with anti-uPA antibody (1:200) followed by <t>HRP-conjugated</t> secondary antibody (1:500). The slides were treated with DAB, counterstained with hematoxylin, and observed under x25 objective. Representative images of each treatment group are shown. (F) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, cells were transferred to transwell chambers coated with matrigel (1 mg/ml) and allowed to invade for 24 hours. Percentage of invaded cells in each treatment group was calculated and plotted. Each blot/image is representative of three independent experiments, and each column represents a mean of three values (asterisk represents significant value of P
    Hrp Conjugated Secondary Antibodies, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 5020 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology horseradish peroxidase hrp conjugated secondary antibody
    PepE target the <t>TrxR1</t> through binding its Sec residue. (A) Inhibition assay of purified recombinant human TrxR1 or TrxR1 without Sec498 residue (nSec498, 0.1 μM) by PepE. The NADPH-reduced proteins were incubated with the indicated concentrations of PepE, and the enzyme's activity was determined by the DTNB assay (Data are presented as mean ± SD, n = 6). (B) Molecular docking of PepE with TrxR1 was carried out using the covalent docking protocol in the Schrodinger Suite. The yellow line indicated the covalent carbon bond, the green dotted lines indicate the hydrogen bonds. (C) Comparison of the binding activity between PepE and TrxR1 protein (red line), PepE and TrxR1 nSec498 protein (blue line), and PepA and TrxR1 protein (black line) through BLI analysis. (D) Kinetic analysis of the interaction between NADPH reduced TrxR1 (0.9 μM) or NADPH reduced TrxR1 nSec498 (0.9 μM) protein and PepE by BLI. The Super Streptavidin (SSA) biosensor tips coated with proteins (0.9 μM) were dipped in increasing concentrations of PepE (0.625, 1.25, 2.5, 5 and 10 μM) to measure the binding affinity of PepE to the proteins (K on ) and subsequently moved to wells containing buffer to measure dissociation rates (K dis ). The affinity constant (K D ) was calculated as the ratio of the K dis to the K on. (E) Kinetic analysis of the interaction between TrxR1 (0.9 μM) and different concentrations of PepA (1.25, 2.5, 5, 10 and 20 μM) by BLI. (F) The interaction of the PepE with the Sec residue in the C -terminal active center of TrxR1. <t>HRP-conjugated</t> streptavidin (HRP-Strep) detection of BIAM labeling of free Selenol in TrxR1 enzyme at pH 6.5 after this enzyme was incubated with different concentrations of PepE (1, 5 and 10 μM), DMSO(negative control), and DNCB (positive control, 5 μM), ** P
    Horseradish Peroxidase Hrp Conjugated Secondary Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 1725 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology horseradish peroxidase hrp
    PepE target the <t>TrxR1</t> through binding its Sec residue. (A) Inhibition assay of purified recombinant human TrxR1 or TrxR1 without Sec498 residue (nSec498, 0.1 μM) by PepE. The NADPH-reduced proteins were incubated with the indicated concentrations of PepE, and the enzyme's activity was determined by the DTNB assay (Data are presented as mean ± SD, n = 6). (B) Molecular docking of PepE with TrxR1 was carried out using the covalent docking protocol in the Schrodinger Suite. The yellow line indicated the covalent carbon bond, the green dotted lines indicate the hydrogen bonds. (C) Comparison of the binding activity between PepE and TrxR1 protein (red line), PepE and TrxR1 nSec498 protein (blue line), and PepA and TrxR1 protein (black line) through BLI analysis. (D) Kinetic analysis of the interaction between NADPH reduced TrxR1 (0.9 μM) or NADPH reduced TrxR1 nSec498 (0.9 μM) protein and PepE by BLI. The Super Streptavidin (SSA) biosensor tips coated with proteins (0.9 μM) were dipped in increasing concentrations of PepE (0.625, 1.25, 2.5, 5 and 10 μM) to measure the binding affinity of PepE to the proteins (K on ) and subsequently moved to wells containing buffer to measure dissociation rates (K dis ). The affinity constant (K D ) was calculated as the ratio of the K dis to the K on. (E) Kinetic analysis of the interaction between TrxR1 (0.9 μM) and different concentrations of PepA (1.25, 2.5, 5, 10 and 20 μM) by BLI. (F) The interaction of the PepE with the Sec residue in the C -terminal active center of TrxR1. <t>HRP-conjugated</t> streptavidin (HRP-Strep) detection of BIAM labeling of free Selenol in TrxR1 enzyme at pH 6.5 after this enzyme was incubated with different concentrations of PepE (1, 5 and 10 μM), DMSO(negative control), and DNCB (positive control, 5 μM), ** P
    Horseradish Peroxidase Hrp, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 2165 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology hrp conjugated goat anti rabbit secondary antibodies
    Induction of γH2AX accumulation by <t>rotavirus</t> infection. Sp2/O-Ag14 cells were infected with tumor cell-adapted rotavirus isolates WTEW, TRUY, WT1-5, WWM or ECwt at MOI of 0.8 and harvested at 12 h.p.i. (A) Immunocytochemistry analysis of infected cells using primary Abs against rotavirus SP and <t>HRP-conjugated</t> goat anti-rabbit secondary antibodies. For phospho-histone H2A.X analysis, cells were permeabilized with 0.5% Triton X-100 and blocked with 3% BSA in TBS buffer. Cells were treated first with anti-phospho-histone H2A.X (Ser 139) mAb (2 μg/ml) and then with FITC-conjugated goat anti-mouse secondary Ab (2 μg/ml). Uninfected cells treated or not with H 2 O 2 (1 mM) were used as a control. (B) Comparison of the mean percentages of cells being positive to rotaviral antigen and γH2AX foci.
    Hrp Conjugated Goat Anti Rabbit Secondary Antibodies, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 119 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti rabbit hrp conjugated secondary antibodies
    Induction of γH2AX accumulation by <t>rotavirus</t> infection. Sp2/O-Ag14 cells were infected with tumor cell-adapted rotavirus isolates WTEW, TRUY, WT1-5, WWM or ECwt at MOI of 0.8 and harvested at 12 h.p.i. (A) Immunocytochemistry analysis of infected cells using primary Abs against rotavirus SP and <t>HRP-conjugated</t> goat anti-rabbit secondary antibodies. For phospho-histone H2A.X analysis, cells were permeabilized with 0.5% Triton X-100 and blocked with 3% BSA in TBS buffer. Cells were treated first with anti-phospho-histone H2A.X (Ser 139) mAb (2 μg/ml) and then with FITC-conjugated goat anti-mouse secondary Ab (2 μg/ml). Uninfected cells treated or not with H 2 O 2 (1 mM) were used as a control. (B) Comparison of the mean percentages of cells being positive to rotaviral antigen and γH2AX foci.
    Anti Rabbit Hrp Conjugated Secondary Antibodies, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 154 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology hrp
    Induction of γH2AX accumulation by <t>rotavirus</t> infection. Sp2/O-Ag14 cells were infected with tumor cell-adapted rotavirus isolates WTEW, TRUY, WT1-5, WWM or ECwt at MOI of 0.8 and harvested at 12 h.p.i. (A) Immunocytochemistry analysis of infected cells using primary Abs against rotavirus SP and <t>HRP-conjugated</t> goat anti-rabbit secondary antibodies. For phospho-histone H2A.X analysis, cells were permeabilized with 0.5% Triton X-100 and blocked with 3% BSA in TBS buffer. Cells were treated first with anti-phospho-histone H2A.X (Ser 139) mAb (2 μg/ml) and then with FITC-conjugated goat anti-mouse secondary Ab (2 μg/ml). Uninfected cells treated or not with H 2 O 2 (1 mM) were used as a control. (B) Comparison of the mean percentages of cells being positive to rotaviral antigen and γH2AX foci.
    Hrp, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1722 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology horseradish peroxidase
    Induction of γH2AX accumulation by <t>rotavirus</t> infection. Sp2/O-Ag14 cells were infected with tumor cell-adapted rotavirus isolates WTEW, TRUY, WT1-5, WWM or ECwt at MOI of 0.8 and harvested at 12 h.p.i. (A) Immunocytochemistry analysis of infected cells using primary Abs against rotavirus SP and <t>HRP-conjugated</t> goat anti-rabbit secondary antibodies. For phospho-histone H2A.X analysis, cells were permeabilized with 0.5% Triton X-100 and blocked with 3% BSA in TBS buffer. Cells were treated first with anti-phospho-histone H2A.X (Ser 139) mAb (2 μg/ml) and then with FITC-conjugated goat anti-mouse secondary Ab (2 μg/ml). Uninfected cells treated or not with H 2 O 2 (1 mM) were used as a control. (B) Comparison of the mean percentages of cells being positive to rotaviral antigen and γH2AX foci.
    Horseradish Peroxidase, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 12982 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology anti mouse hrp conjugated secondary antibody
    Induction of γH2AX accumulation by <t>rotavirus</t> infection. Sp2/O-Ag14 cells were infected with tumor cell-adapted rotavirus isolates WTEW, TRUY, WT1-5, WWM or ECwt at MOI of 0.8 and harvested at 12 h.p.i. (A) Immunocytochemistry analysis of infected cells using primary Abs against rotavirus SP and <t>HRP-conjugated</t> goat anti-rabbit secondary antibodies. For phospho-histone H2A.X analysis, cells were permeabilized with 0.5% Triton X-100 and blocked with 3% BSA in TBS buffer. Cells were treated first with anti-phospho-histone H2A.X (Ser 139) mAb (2 μg/ml) and then with FITC-conjugated goat anti-mouse secondary Ab (2 μg/ml). Uninfected cells treated or not with H 2 O 2 (1 mM) were used as a control. (B) Comparison of the mean percentages of cells being positive to rotaviral antigen and γH2AX foci.
    Anti Mouse Hrp Conjugated Secondary Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 198 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Santa Cruz Biotechnology goat anti rabbit igg hrp conjugated secondary antibody
    Induction of γH2AX accumulation by <t>rotavirus</t> infection. Sp2/O-Ag14 cells were infected with tumor cell-adapted rotavirus isolates WTEW, TRUY, WT1-5, WWM or ECwt at MOI of 0.8 and harvested at 12 h.p.i. (A) Immunocytochemistry analysis of infected cells using primary Abs against rotavirus SP and <t>HRP-conjugated</t> goat anti-rabbit secondary antibodies. For phospho-histone H2A.X analysis, cells were permeabilized with 0.5% Triton X-100 and blocked with 3% BSA in TBS buffer. Cells were treated first with anti-phospho-histone H2A.X (Ser 139) mAb (2 μg/ml) and then with FITC-conjugated goat anti-mouse secondary Ab (2 μg/ml). Uninfected cells treated or not with H 2 O 2 (1 mM) were used as a control. (B) Comparison of the mean percentages of cells being positive to rotaviral antigen and γH2AX foci.
    Goat Anti Rabbit Igg Hrp Conjugated Secondary Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 152 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology hrp conjugated goat anti mouse secondary antibody
    Application of the <t>anti–Rae-1</t> mAb in Western blotting and flow cytometry. A . Detection of Rae-1 recombinant protein in Western blotting using the 52A mAb. Rae-1b recombinant protein (20 μg and 5 μg) and control human IgG-Fc (20 μg) were loaded onto 10% sodium dodecyl sulfate–polyacrylamide gel. In the Western blot assay, the primary antibody was the 52A anti–Rae-1 mAb, and the secondary antibody was <t>HRP</t> goat anti-mouse IgG. B . Detection of Rae-1 expression levels in multiple murine cancer cell lines using the 52A anti–Rae-1 mAb. Murine tumor cells were not stained, stained with isotype control, or stained with the 52A anti–Rae-1 mAb. Rae-1 expression levels were assessed using flow cytometry. a , K7M3 cells; b , LLC cells; c , TC1 cells; d , CT26 cells; e , B16F10 cells.
    Hrp Conjugated Goat Anti Mouse Secondary Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 235 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology secondary ab conjugated to hrp
    Activated T cells possess active full-length <t>caspase-8,</t> active cleaved caspase-3, full-length FLIP L , p43FLIP, and RIP1 complexed with active caspases. Freshly isolated polyclonal T cells ( lanes 1 and 3 ) or day 4 effectors ( lanes 2 , 4 , and 5 ) were incubated with control peptide z-FA-fmk ( lanes 1 - 4 ) or 100 μ M nonbiotinylated z-VAD ( lane 5 ) at 37°C for 15 min. All cells were then incubated with 10 μ M biotin-VAD-fmk for an additional 15 min at 37°C. Subsequently, cells were lysed in buffer containing 20 μ M biotin-VAD ( lanes 1 - 5 ). A portion of lysates were then incubated with avidin-Sepharose beads to precipitate active caspases. Immunoblot analysis was performed on precipitates for caspase-8 ( top panel ), caspase-3 ( middle panel ), or RIP1 ( bottom panel ) to reveal active caspases and RIP1 cleavage products. As a positive control for the presence and size of caspase-8, caspase-3, and RIP1, a portion of nonprecipitated whole cell lysates (WCLs) was included in the analysis from fresh ( lane 1 ) or day 4 effectors ( lane 2 ). B , Active caspases were precipitated from day 4 effector lysates and analyzed by immunoblot for the indicated molecules. WCLs were included as a reference comparison. A longer exposure of the caspase-8 immunoblot is included to illustrate precipitation of p43/41 caspase-8. C , Day 4 effectors were treated with FasL for 1 or 2 h, followed by biotin-VAD for 15 min. Active caspases were then precipitated and immunoblot analysis was performed for caspase-8. A portion of the fraction not binding to avidin-Sepharose (Flow Through) as well as WCLs were included in analysis. Bands were revealed using anti-caspase-8 Ab ( top panel ) or <t>HRP-conjugated</t> Strepavidin ( middle and bottom panels ). Bands at 55 and 43/41 kDa were identified, which corresponds with the bands revealed in the caspase-8 immunoblots ( top and middle panels ). In addition, a band of 17 kDa was observed that corresponds with cleaved p17 caspase-3 ( bottom panel ).
    Secondary Ab Conjugated To Hrp, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Santa Cruz Biotechnology goat anti mouse igg horseradish peroxidase conjugated secondary antibody
    Activated T cells possess active full-length <t>caspase-8,</t> active cleaved caspase-3, full-length FLIP L , p43FLIP, and RIP1 complexed with active caspases. Freshly isolated polyclonal T cells ( lanes 1 and 3 ) or day 4 effectors ( lanes 2 , 4 , and 5 ) were incubated with control peptide z-FA-fmk ( lanes 1 - 4 ) or 100 μ M nonbiotinylated z-VAD ( lane 5 ) at 37°C for 15 min. All cells were then incubated with 10 μ M biotin-VAD-fmk for an additional 15 min at 37°C. Subsequently, cells were lysed in buffer containing 20 μ M biotin-VAD ( lanes 1 - 5 ). A portion of lysates were then incubated with avidin-Sepharose beads to precipitate active caspases. Immunoblot analysis was performed on precipitates for caspase-8 ( top panel ), caspase-3 ( middle panel ), or RIP1 ( bottom panel ) to reveal active caspases and RIP1 cleavage products. As a positive control for the presence and size of caspase-8, caspase-3, and RIP1, a portion of nonprecipitated whole cell lysates (WCLs) was included in the analysis from fresh ( lane 1 ) or day 4 effectors ( lane 2 ). B , Active caspases were precipitated from day 4 effector lysates and analyzed by immunoblot for the indicated molecules. WCLs were included as a reference comparison. A longer exposure of the caspase-8 immunoblot is included to illustrate precipitation of p43/41 caspase-8. C , Day 4 effectors were treated with FasL for 1 or 2 h, followed by biotin-VAD for 15 min. Active caspases were then precipitated and immunoblot analysis was performed for caspase-8. A portion of the fraction not binding to avidin-Sepharose (Flow Through) as well as WCLs were included in analysis. Bands were revealed using anti-caspase-8 Ab ( top panel ) or <t>HRP-conjugated</t> Strepavidin ( middle and bottom panels ). Bands at 55 and 43/41 kDa were identified, which corresponds with the bands revealed in the caspase-8 immunoblots ( top and middle panels ). In addition, a band of 17 kDa was observed that corresponds with cleaved p17 caspase-3 ( bottom panel ).
    Goat Anti Mouse Igg Horseradish Peroxidase Conjugated Secondary Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Santa Cruz Biotechnology horseradish peroxidase hrp conjugated goat anti rabbit secondary antibody
    Activated T cells possess active full-length <t>caspase-8,</t> active cleaved caspase-3, full-length FLIP L , p43FLIP, and RIP1 complexed with active caspases. Freshly isolated polyclonal T cells ( lanes 1 and 3 ) or day 4 effectors ( lanes 2 , 4 , and 5 ) were incubated with control peptide z-FA-fmk ( lanes 1 - 4 ) or 100 μ M nonbiotinylated z-VAD ( lane 5 ) at 37°C for 15 min. All cells were then incubated with 10 μ M biotin-VAD-fmk for an additional 15 min at 37°C. Subsequently, cells were lysed in buffer containing 20 μ M biotin-VAD ( lanes 1 - 5 ). A portion of lysates were then incubated with avidin-Sepharose beads to precipitate active caspases. Immunoblot analysis was performed on precipitates for caspase-8 ( top panel ), caspase-3 ( middle panel ), or RIP1 ( bottom panel ) to reveal active caspases and RIP1 cleavage products. As a positive control for the presence and size of caspase-8, caspase-3, and RIP1, a portion of nonprecipitated whole cell lysates (WCLs) was included in the analysis from fresh ( lane 1 ) or day 4 effectors ( lane 2 ). B , Active caspases were precipitated from day 4 effector lysates and analyzed by immunoblot for the indicated molecules. WCLs were included as a reference comparison. A longer exposure of the caspase-8 immunoblot is included to illustrate precipitation of p43/41 caspase-8. C , Day 4 effectors were treated with FasL for 1 or 2 h, followed by biotin-VAD for 15 min. Active caspases were then precipitated and immunoblot analysis was performed for caspase-8. A portion of the fraction not binding to avidin-Sepharose (Flow Through) as well as WCLs were included in analysis. Bands were revealed using anti-caspase-8 Ab ( top panel ) or <t>HRP-conjugated</t> Strepavidin ( middle and bottom panels ). Bands at 55 and 43/41 kDa were identified, which corresponds with the bands revealed in the caspase-8 immunoblots ( top and middle panels ). In addition, a band of 17 kDa was observed that corresponds with cleaved p17 caspase-3 ( bottom panel ).
    Horseradish Peroxidase Hrp Conjugated Goat Anti Rabbit Secondary Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 113 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology hrp conjugated goat anti mouse igg secondary antibody
    Activated T cells possess active full-length <t>caspase-8,</t> active cleaved caspase-3, full-length FLIP L , p43FLIP, and RIP1 complexed with active caspases. Freshly isolated polyclonal T cells ( lanes 1 and 3 ) or day 4 effectors ( lanes 2 , 4 , and 5 ) were incubated with control peptide z-FA-fmk ( lanes 1 - 4 ) or 100 μ M nonbiotinylated z-VAD ( lane 5 ) at 37°C for 15 min. All cells were then incubated with 10 μ M biotin-VAD-fmk for an additional 15 min at 37°C. Subsequently, cells were lysed in buffer containing 20 μ M biotin-VAD ( lanes 1 - 5 ). A portion of lysates were then incubated with avidin-Sepharose beads to precipitate active caspases. Immunoblot analysis was performed on precipitates for caspase-8 ( top panel ), caspase-3 ( middle panel ), or RIP1 ( bottom panel ) to reveal active caspases and RIP1 cleavage products. As a positive control for the presence and size of caspase-8, caspase-3, and RIP1, a portion of nonprecipitated whole cell lysates (WCLs) was included in the analysis from fresh ( lane 1 ) or day 4 effectors ( lane 2 ). B , Active caspases were precipitated from day 4 effector lysates and analyzed by immunoblot for the indicated molecules. WCLs were included as a reference comparison. A longer exposure of the caspase-8 immunoblot is included to illustrate precipitation of p43/41 caspase-8. C , Day 4 effectors were treated with FasL for 1 or 2 h, followed by biotin-VAD for 15 min. Active caspases were then precipitated and immunoblot analysis was performed for caspase-8. A portion of the fraction not binding to avidin-Sepharose (Flow Through) as well as WCLs were included in analysis. Bands were revealed using anti-caspase-8 Ab ( top panel ) or <t>HRP-conjugated</t> Strepavidin ( middle and bottom panels ). Bands at 55 and 43/41 kDa were identified, which corresponds with the bands revealed in the caspase-8 immunoblots ( top and middle panels ). In addition, a band of 17 kDa was observed that corresponds with cleaved p17 caspase-3 ( bottom panel ).
    Hrp Conjugated Goat Anti Mouse Igg Secondary Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 94 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    siRNA directed toward the uPA promoter prevents radiation-induced hypomethylation. (A) Schematic depiction of the uPA promoter showing shRNA target region, Sp1 binding sites, and the region selected for MSP. (B) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, bisulfite-treated genomic DNA was subjected to MSP and followed by agarose gel analysis. (C) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, conditioned media were subjected to fibrin zymography, total RNA was used to perform RT-PCR, and cell lysates were used for immunoblot analysis as indicated. (D) The band intensities were quantified and represented as arbitrary units. (E) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, cells were fixed and subjected to immunocytochemistry with anti-uPA antibody (1:200) followed by HRP-conjugated secondary antibody (1:500). The slides were treated with DAB, counterstained with hematoxylin, and observed under x25 objective. Representative images of each treatment group are shown. (F) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, cells were transferred to transwell chambers coated with matrigel (1 mg/ml) and allowed to invade for 24 hours. Percentage of invaded cells in each treatment group was calculated and plotted. Each blot/image is representative of three independent experiments, and each column represents a mean of three values (asterisk represents significant value of P

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Radiation-Induced Hypomethylation Triggers Urokinase Plasminogen Activator Transcription in Meningioma Cells 1

    doi:

    Figure Lengend Snippet: siRNA directed toward the uPA promoter prevents radiation-induced hypomethylation. (A) Schematic depiction of the uPA promoter showing shRNA target region, Sp1 binding sites, and the region selected for MSP. (B) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, bisulfite-treated genomic DNA was subjected to MSP and followed by agarose gel analysis. (C) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, conditioned media were subjected to fibrin zymography, total RNA was used to perform RT-PCR, and cell lysates were used for immunoblot analysis as indicated. (D) The band intensities were quantified and represented as arbitrary units. (E) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, cells were fixed and subjected to immunocytochemistry with anti-uPA antibody (1:200) followed by HRP-conjugated secondary antibody (1:500). The slides were treated with DAB, counterstained with hematoxylin, and observed under x25 objective. Representative images of each treatment group are shown. (F) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, cells were transferred to transwell chambers coated with matrigel (1 mg/ml) and allowed to invade for 24 hours. Percentage of invaded cells in each treatment group was calculated and plotted. Each blot/image is representative of three independent experiments, and each column represents a mean of three values (asterisk represents significant value of P

    Article Snippet: DNMT1, phospho extracellular signal-regulated kinase (pERK), ERK, MEK, phospho MAP kinase kinase (pMEK), cRaf, pcRAF, uPA, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies and HRP-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: shRNA, Binding Assay, Transfection, Irradiation, Incubation, Agarose Gel Electrophoresis, Zymography, Reverse Transcription Polymerase Chain Reaction, Immunocytochemistry

    Radiation treatment induces hypomethylation in vivo . Nude mice were intracranially implanted with IOMM-Lee cells as described in the Materials and Methods section. (A) Formalin-fixed brain sections were subjected to H E staining and observed under x20 objective. Each image represents a brain section of five animals. (B) Total RNA was extracted from frozen brain tissues, and RT-PCR was performed for uPA and GAPDH. Tissue lysates were analyzed for uPA expression by Western blot analysis. (C) The band intensities of RT-PCR gels and Western blots were quantified and represented as arbitrary units. (D) Genomic DNA from frozen brain tissues was extracted, treated with bisulfite, and subjected to MSP targeting the uPA promoter (MP, methylated primer; UMP, unmethylated primer). (E) The band intensities were quantified and represented as arbitrary units. (F) Immunostaining with anti-DNMT1 antibody and non-specific IgG (NsIgG) was performed on formalin-fixed brain sections from animals implanted with IOMM-Lee cells followed by treatment with HRP-conjugated secondary antibody and DAB staining. Representative photomicrographs (x200) are shown in comparison to normal mouse brain sections. (G) Immunohistochemical analysis for DNMT1 was performed on tissue microarrays and clinical meningioma samples. Representative images (x400) are shown. Each column represents a mean value of brain tissues from three different animals (* P

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Radiation-Induced Hypomethylation Triggers Urokinase Plasminogen Activator Transcription in Meningioma Cells 1

    doi:

    Figure Lengend Snippet: Radiation treatment induces hypomethylation in vivo . Nude mice were intracranially implanted with IOMM-Lee cells as described in the Materials and Methods section. (A) Formalin-fixed brain sections were subjected to H E staining and observed under x20 objective. Each image represents a brain section of five animals. (B) Total RNA was extracted from frozen brain tissues, and RT-PCR was performed for uPA and GAPDH. Tissue lysates were analyzed for uPA expression by Western blot analysis. (C) The band intensities of RT-PCR gels and Western blots were quantified and represented as arbitrary units. (D) Genomic DNA from frozen brain tissues was extracted, treated with bisulfite, and subjected to MSP targeting the uPA promoter (MP, methylated primer; UMP, unmethylated primer). (E) The band intensities were quantified and represented as arbitrary units. (F) Immunostaining with anti-DNMT1 antibody and non-specific IgG (NsIgG) was performed on formalin-fixed brain sections from animals implanted with IOMM-Lee cells followed by treatment with HRP-conjugated secondary antibody and DAB staining. Representative photomicrographs (x200) are shown in comparison to normal mouse brain sections. (G) Immunohistochemical analysis for DNMT1 was performed on tissue microarrays and clinical meningioma samples. Representative images (x400) are shown. Each column represents a mean value of brain tissues from three different animals (* P

    Article Snippet: DNMT1, phospho extracellular signal-regulated kinase (pERK), ERK, MEK, phospho MAP kinase kinase (pMEK), cRaf, pcRAF, uPA, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies and HRP-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: In Vivo, Mouse Assay, Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Methylation, Immunostaining, Immunohistochemistry

    PepE target the TrxR1 through binding its Sec residue. (A) Inhibition assay of purified recombinant human TrxR1 or TrxR1 without Sec498 residue (nSec498, 0.1 μM) by PepE. The NADPH-reduced proteins were incubated with the indicated concentrations of PepE, and the enzyme's activity was determined by the DTNB assay (Data are presented as mean ± SD, n = 6). (B) Molecular docking of PepE with TrxR1 was carried out using the covalent docking protocol in the Schrodinger Suite. The yellow line indicated the covalent carbon bond, the green dotted lines indicate the hydrogen bonds. (C) Comparison of the binding activity between PepE and TrxR1 protein (red line), PepE and TrxR1 nSec498 protein (blue line), and PepA and TrxR1 protein (black line) through BLI analysis. (D) Kinetic analysis of the interaction between NADPH reduced TrxR1 (0.9 μM) or NADPH reduced TrxR1 nSec498 (0.9 μM) protein and PepE by BLI. The Super Streptavidin (SSA) biosensor tips coated with proteins (0.9 μM) were dipped in increasing concentrations of PepE (0.625, 1.25, 2.5, 5 and 10 μM) to measure the binding affinity of PepE to the proteins (K on ) and subsequently moved to wells containing buffer to measure dissociation rates (K dis ). The affinity constant (K D ) was calculated as the ratio of the K dis to the K on. (E) Kinetic analysis of the interaction between TrxR1 (0.9 μM) and different concentrations of PepA (1.25, 2.5, 5, 10 and 20 μM) by BLI. (F) The interaction of the PepE with the Sec residue in the C -terminal active center of TrxR1. HRP-conjugated streptavidin (HRP-Strep) detection of BIAM labeling of free Selenol in TrxR1 enzyme at pH 6.5 after this enzyme was incubated with different concentrations of PepE (1, 5 and 10 μM), DMSO(negative control), and DNCB (positive control, 5 μM), ** P

    Journal: Redox Biology

    Article Title: Peperomin E and its orally bioavailable analog induce oxidative stress-mediated apoptosis of acute myeloid leukemia progenitor cells by targeting thioredoxin reductase

    doi: 10.1016/j.redox.2019.101153

    Figure Lengend Snippet: PepE target the TrxR1 through binding its Sec residue. (A) Inhibition assay of purified recombinant human TrxR1 or TrxR1 without Sec498 residue (nSec498, 0.1 μM) by PepE. The NADPH-reduced proteins were incubated with the indicated concentrations of PepE, and the enzyme's activity was determined by the DTNB assay (Data are presented as mean ± SD, n = 6). (B) Molecular docking of PepE with TrxR1 was carried out using the covalent docking protocol in the Schrodinger Suite. The yellow line indicated the covalent carbon bond, the green dotted lines indicate the hydrogen bonds. (C) Comparison of the binding activity between PepE and TrxR1 protein (red line), PepE and TrxR1 nSec498 protein (blue line), and PepA and TrxR1 protein (black line) through BLI analysis. (D) Kinetic analysis of the interaction between NADPH reduced TrxR1 (0.9 μM) or NADPH reduced TrxR1 nSec498 (0.9 μM) protein and PepE by BLI. The Super Streptavidin (SSA) biosensor tips coated with proteins (0.9 μM) were dipped in increasing concentrations of PepE (0.625, 1.25, 2.5, 5 and 10 μM) to measure the binding affinity of PepE to the proteins (K on ) and subsequently moved to wells containing buffer to measure dissociation rates (K dis ). The affinity constant (K D ) was calculated as the ratio of the K dis to the K on. (E) Kinetic analysis of the interaction between TrxR1 (0.9 μM) and different concentrations of PepA (1.25, 2.5, 5, 10 and 20 μM) by BLI. (F) The interaction of the PepE with the Sec residue in the C -terminal active center of TrxR1. HRP-conjugated streptavidin (HRP-Strep) detection of BIAM labeling of free Selenol in TrxR1 enzyme at pH 6.5 after this enzyme was incubated with different concentrations of PepE (1, 5 and 10 μM), DMSO(negative control), and DNCB (positive control, 5 μM), ** P

    Article Snippet: Horseradish peroxidase (HRP)-conjugated secondary antibody (goat anti-rabbit), human TrxR1 shRNA plasmids, control shRNA plasmids, TrxR1 CRISPR activation plasmids, control CRISPR activation plasmids were obtained from Santa Cruz Biotech.

    Techniques: Binding Assay, Size-exclusion Chromatography, Inhibition, Purification, Recombinant, Incubation, Activity Assay, DTNB Assay, Labeling, Positive Control

    Induction of γH2AX accumulation by rotavirus infection. Sp2/O-Ag14 cells were infected with tumor cell-adapted rotavirus isolates WTEW, TRUY, WT1-5, WWM or ECwt at MOI of 0.8 and harvested at 12 h.p.i. (A) Immunocytochemistry analysis of infected cells using primary Abs against rotavirus SP and HRP-conjugated goat anti-rabbit secondary antibodies. For phospho-histone H2A.X analysis, cells were permeabilized with 0.5% Triton X-100 and blocked with 3% BSA in TBS buffer. Cells were treated first with anti-phospho-histone H2A.X (Ser 139) mAb (2 μg/ml) and then with FITC-conjugated goat anti-mouse secondary Ab (2 μg/ml). Uninfected cells treated or not with H 2 O 2 (1 mM) were used as a control. (B) Comparison of the mean percentages of cells being positive to rotaviral antigen and γH2AX foci.

    Journal: PLoS ONE

    Article Title: Experimental Adaptation of Rotaviruses to Tumor Cell Lines

    doi: 10.1371/journal.pone.0147666

    Figure Lengend Snippet: Induction of γH2AX accumulation by rotavirus infection. Sp2/O-Ag14 cells were infected with tumor cell-adapted rotavirus isolates WTEW, TRUY, WT1-5, WWM or ECwt at MOI of 0.8 and harvested at 12 h.p.i. (A) Immunocytochemistry analysis of infected cells using primary Abs against rotavirus SP and HRP-conjugated goat anti-rabbit secondary antibodies. For phospho-histone H2A.X analysis, cells were permeabilized with 0.5% Triton X-100 and blocked with 3% BSA in TBS buffer. Cells were treated first with anti-phospho-histone H2A.X (Ser 139) mAb (2 μg/ml) and then with FITC-conjugated goat anti-mouse secondary Ab (2 μg/ml). Uninfected cells treated or not with H 2 O 2 (1 mM) were used as a control. (B) Comparison of the mean percentages of cells being positive to rotaviral antigen and γH2AX foci.

    Article Snippet: The infection was determined by immunochemistry using polyclonal rabbit Abs (1:2000) against rotavirus structural proteins and HRP-conjugated goat anti-rabbit secondary antibodies (0.13 μg/ml, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

    Techniques: Infection, Immunocytochemistry

    Identification of AKAPs in dendritic cells by western analysis. Monocytes were isolated from PBMC by negative isolation of CD14 + cells (Mo), plated in culture for 5 days with GM-CSF and IL-4 (immature DC; iDC) or plated for five days with GM-CSF and IL-4 and an additional two days with LPS (mature DC; mDC). See Materials and Methods for details. A) One representative set of westerns in which 10 µg total protein was loaded in each lane. Membranes were probed with monoclonal antibodies to AKAP149, AKAP95, AKAP79, RIIα, RIα, and GAPDH, goat-anti-mouse HRP conjugated secondary and chemiluminescence detection. B) One representative western in which 10 µg of total protein was loaded in each lane. The membrane was probed with a rabbit polyclonal antibody to AKAP-Lbc (generous gift of Dr. Diviani), goat-anti-rabbit HRP conjugated secondary and chemiluminescence detection. C) Quantitation of five westerns using NIH image quantitation software. One way ANOVA for three correlated samples yielded significance with p = 0.0004 for AKAP149; p

    Journal: PLoS ONE

    Article Title: A-Kinase Anchoring in Dendritic Cells Is Required for Antigen Presentation

    doi: 10.1371/journal.pone.0004807

    Figure Lengend Snippet: Identification of AKAPs in dendritic cells by western analysis. Monocytes were isolated from PBMC by negative isolation of CD14 + cells (Mo), plated in culture for 5 days with GM-CSF and IL-4 (immature DC; iDC) or plated for five days with GM-CSF and IL-4 and an additional two days with LPS (mature DC; mDC). See Materials and Methods for details. A) One representative set of westerns in which 10 µg total protein was loaded in each lane. Membranes were probed with monoclonal antibodies to AKAP149, AKAP95, AKAP79, RIIα, RIα, and GAPDH, goat-anti-mouse HRP conjugated secondary and chemiluminescence detection. B) One representative western in which 10 µg of total protein was loaded in each lane. The membrane was probed with a rabbit polyclonal antibody to AKAP-Lbc (generous gift of Dr. Diviani), goat-anti-rabbit HRP conjugated secondary and chemiluminescence detection. C) Quantitation of five westerns using NIH image quantitation software. One way ANOVA for three correlated samples yielded significance with p = 0.0004 for AKAP149; p

    Article Snippet: Membranes were blocked in 5% milk-TTBS for 1 hour, incubated with anti-AKAP-Lbc antibody (generously provided by Dr. Dario Diviani) at 1∶1000 dilution, and goat-anti-rabbit HRP conjugated secondary antibody at 1∶5000 dilution (Santa Cruz biotechnology).

    Techniques: Western Blot, Isolation, Quantitation Assay, Software

    Application of the anti–Rae-1 mAb in Western blotting and flow cytometry. A . Detection of Rae-1 recombinant protein in Western blotting using the 52A mAb. Rae-1b recombinant protein (20 μg and 5 μg) and control human IgG-Fc (20 μg) were loaded onto 10% sodium dodecyl sulfate–polyacrylamide gel. In the Western blot assay, the primary antibody was the 52A anti–Rae-1 mAb, and the secondary antibody was HRP goat anti-mouse IgG. B . Detection of Rae-1 expression levels in multiple murine cancer cell lines using the 52A anti–Rae-1 mAb. Murine tumor cells were not stained, stained with isotype control, or stained with the 52A anti–Rae-1 mAb. Rae-1 expression levels were assessed using flow cytometry. a , K7M3 cells; b , LLC cells; c , TC1 cells; d , CT26 cells; e , B16F10 cells.

    Journal: Biological Procedures Online

    Article Title: Generation of a monoclonal antibody against the glycosylphosphatidylinositol-linked protein Rae-1 using genetically engineered tumor cells

    doi: 10.1186/1480-9222-16-3

    Figure Lengend Snippet: Application of the anti–Rae-1 mAb in Western blotting and flow cytometry. A . Detection of Rae-1 recombinant protein in Western blotting using the 52A mAb. Rae-1b recombinant protein (20 μg and 5 μg) and control human IgG-Fc (20 μg) were loaded onto 10% sodium dodecyl sulfate–polyacrylamide gel. In the Western blot assay, the primary antibody was the 52A anti–Rae-1 mAb, and the secondary antibody was HRP goat anti-mouse IgG. B . Detection of Rae-1 expression levels in multiple murine cancer cell lines using the 52A anti–Rae-1 mAb. Murine tumor cells were not stained, stained with isotype control, or stained with the 52A anti–Rae-1 mAb. Rae-1 expression levels were assessed using flow cytometry. a , K7M3 cells; b , LLC cells; c , TC1 cells; d , CT26 cells; e , B16F10 cells.

    Article Snippet: The membranes were blotted with anit–Rae-1 primary antibody and HRP-conjugated goat anti-mouse secondary antibody (Santa Cruz Biotechnology, Dallas, TX) to detect the protein of interest.

    Techniques: Western Blot, Flow Cytometry, Cytometry, Recombinant, Expressing, Staining

    Application of the anti–Rae-1 mAb in immunohistochemistry staining of tissue sections. CT26–Rae-1 and CT26-GFP frozen tumor sections were stained with the 52A anti–Rae-1 mAb and then stained with HRP-conjugated goat anti-mouse secondary antibody or secondary antibody alone.

    Journal: Biological Procedures Online

    Article Title: Generation of a monoclonal antibody against the glycosylphosphatidylinositol-linked protein Rae-1 using genetically engineered tumor cells

    doi: 10.1186/1480-9222-16-3

    Figure Lengend Snippet: Application of the anti–Rae-1 mAb in immunohistochemistry staining of tissue sections. CT26–Rae-1 and CT26-GFP frozen tumor sections were stained with the 52A anti–Rae-1 mAb and then stained with HRP-conjugated goat anti-mouse secondary antibody or secondary antibody alone.

    Article Snippet: The membranes were blotted with anit–Rae-1 primary antibody and HRP-conjugated goat anti-mouse secondary antibody (Santa Cruz Biotechnology, Dallas, TX) to detect the protein of interest.

    Techniques: Immunohistochemistry, Staining

    An N-glycosylation analysis of the fE1E2 and tE1E2 complex expressed in L. tarentolae . ( A ) The recombinant complexes were treated with endoglycosidase PNGaseF. After overnight incubation at 37 °C in native conditions, western blot under reducing conditions with anti-E2 Ab was performed. The E1E2 complex expressed in HEK 293 cells was used as the control (hE1E2). ( B ) Dose-dependent binding of the E1E2 complexes to GNA lectin. Decreasing concentrations of cell lysates (5-fold dilutions - from 1:5 to 1:3125) were captured in ELISA plates coated with GNA lectin. The bound antigens were visualized with anti-E2 Abs, anti-mouse IgG HRP conjugate, and the TMB substrate. The error bars represent the standard deviations of 2 replicate values.

    Journal: Scientific Reports

    Article Title: Immunogenicity and functional characterization of Leishmania-derived hepatitis C virus envelope glycoprotein complex

    doi: 10.1038/srep30627

    Figure Lengend Snippet: An N-glycosylation analysis of the fE1E2 and tE1E2 complex expressed in L. tarentolae . ( A ) The recombinant complexes were treated with endoglycosidase PNGaseF. After overnight incubation at 37 °C in native conditions, western blot under reducing conditions with anti-E2 Ab was performed. The E1E2 complex expressed in HEK 293 cells was used as the control (hE1E2). ( B ) Dose-dependent binding of the E1E2 complexes to GNA lectin. Decreasing concentrations of cell lysates (5-fold dilutions - from 1:5 to 1:3125) were captured in ELISA plates coated with GNA lectin. The bound antigens were visualized with anti-E2 Abs, anti-mouse IgG HRP conjugate, and the TMB substrate. The error bars represent the standard deviations of 2 replicate values.

    Article Snippet: The binding of the antibodies to the recombinant proteins was detected by goat anti-mouse HRP-conjugated secondary antibodies diluted 1:2000 (Santa Cruz) and the TMB substrate.

    Techniques: Recombinant, Incubation, Western Blot, Binding Assay, Enzyme-linked Immunosorbent Assay

    Activated T cells possess active full-length caspase-8, active cleaved caspase-3, full-length FLIP L , p43FLIP, and RIP1 complexed with active caspases. Freshly isolated polyclonal T cells ( lanes 1 and 3 ) or day 4 effectors ( lanes 2 , 4 , and 5 ) were incubated with control peptide z-FA-fmk ( lanes 1 - 4 ) or 100 μ M nonbiotinylated z-VAD ( lane 5 ) at 37°C for 15 min. All cells were then incubated with 10 μ M biotin-VAD-fmk for an additional 15 min at 37°C. Subsequently, cells were lysed in buffer containing 20 μ M biotin-VAD ( lanes 1 - 5 ). A portion of lysates were then incubated with avidin-Sepharose beads to precipitate active caspases. Immunoblot analysis was performed on precipitates for caspase-8 ( top panel ), caspase-3 ( middle panel ), or RIP1 ( bottom panel ) to reveal active caspases and RIP1 cleavage products. As a positive control for the presence and size of caspase-8, caspase-3, and RIP1, a portion of nonprecipitated whole cell lysates (WCLs) was included in the analysis from fresh ( lane 1 ) or day 4 effectors ( lane 2 ). B , Active caspases were precipitated from day 4 effector lysates and analyzed by immunoblot for the indicated molecules. WCLs were included as a reference comparison. A longer exposure of the caspase-8 immunoblot is included to illustrate precipitation of p43/41 caspase-8. C , Day 4 effectors were treated with FasL for 1 or 2 h, followed by biotin-VAD for 15 min. Active caspases were then precipitated and immunoblot analysis was performed for caspase-8. A portion of the fraction not binding to avidin-Sepharose (Flow Through) as well as WCLs were included in analysis. Bands were revealed using anti-caspase-8 Ab ( top panel ) or HRP-conjugated Strepavidin ( middle and bottom panels ). Bands at 55 and 43/41 kDa were identified, which corresponds with the bands revealed in the caspase-8 immunoblots ( top and middle panels ). In addition, a band of 17 kDa was observed that corresponds with cleaved p17 caspase-3 ( bottom panel ).

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Effector CD4+ T Cells Generate Intermediate Caspase Activity and Cleavage of Caspase-8 Substrates

    doi:

    Figure Lengend Snippet: Activated T cells possess active full-length caspase-8, active cleaved caspase-3, full-length FLIP L , p43FLIP, and RIP1 complexed with active caspases. Freshly isolated polyclonal T cells ( lanes 1 and 3 ) or day 4 effectors ( lanes 2 , 4 , and 5 ) were incubated with control peptide z-FA-fmk ( lanes 1 - 4 ) or 100 μ M nonbiotinylated z-VAD ( lane 5 ) at 37°C for 15 min. All cells were then incubated with 10 μ M biotin-VAD-fmk for an additional 15 min at 37°C. Subsequently, cells were lysed in buffer containing 20 μ M biotin-VAD ( lanes 1 - 5 ). A portion of lysates were then incubated with avidin-Sepharose beads to precipitate active caspases. Immunoblot analysis was performed on precipitates for caspase-8 ( top panel ), caspase-3 ( middle panel ), or RIP1 ( bottom panel ) to reveal active caspases and RIP1 cleavage products. As a positive control for the presence and size of caspase-8, caspase-3, and RIP1, a portion of nonprecipitated whole cell lysates (WCLs) was included in the analysis from fresh ( lane 1 ) or day 4 effectors ( lane 2 ). B , Active caspases were precipitated from day 4 effector lysates and analyzed by immunoblot for the indicated molecules. WCLs were included as a reference comparison. A longer exposure of the caspase-8 immunoblot is included to illustrate precipitation of p43/41 caspase-8. C , Day 4 effectors were treated with FasL for 1 or 2 h, followed by biotin-VAD for 15 min. Active caspases were then precipitated and immunoblot analysis was performed for caspase-8. A portion of the fraction not binding to avidin-Sepharose (Flow Through) as well as WCLs were included in analysis. Bands were revealed using anti-caspase-8 Ab ( top panel ) or HRP-conjugated Strepavidin ( middle and bottom panels ). Bands at 55 and 43/41 kDa were identified, which corresponds with the bands revealed in the caspase-8 immunoblots ( top and middle panels ). In addition, a band of 17 kDa was observed that corresponds with cleaved p17 caspase-3 ( bottom panel ).

    Article Snippet: Blots were washed three times for 15 min each and incubated for 90 min with 0.5 μ g/ml (caspase blots) or 1 μ g/ml (all others) of appropriate secondary Ab conjugated to HRP (Santa Cruz Biotechnology).

    Techniques: Isolation, Incubation, Avidin-Biotin Assay, Positive Control, Binding Assay, Flow Cytometry, Western Blot