goat anti-rabbit igg Thermo Fisher Search Results


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  • 90
    Thermo Fisher alexa 488 conjugated secondary antibody
    JEV infection downregulates miR-432 levels and leads to SOCS5 upregulation. Human brain microglial cells were infected by JEV (MOI 5) and cells were harvested at various time points to determine miR-432 levels. (A) Graph showing downregulation of miR-432 levels at 24 and 48 hours post infection as compared to 12 hours sample. TaqMan probe specific to miR-432 were used to determine fold change. The C t values were normalized by RNU-24 levels. (B) Graph showing reduced levels of miR-432 in JEV infected mice brain. The RNA was isolated from brain tissue of 2 day and 4 day infected mice and mock infected mice brain was used as control. The C t values were normalized by RNU-6 levels. (C) Western blots depicting upregulation of SOCS5 upon JEV infection. CHME3 cells were infected by JEV (MOI 5) and harvested after 24 and 48 hours. The average fold change values with respect to control have been mentioned. (D) Western blots showing upregulation of SOCS5 in JEV infected brain tissue. Mock infected mice brain tissue was used as control. Infected mice were harvested 2 day and 4 day post infection. β-tubulin was used for normalization. The average fold change values with respect to control have been mentioned. (E) Immunohistochemistry image showing increased levels of SOCS5 protein in brain of JEV infected BALB/c mice. Mock and JEV infected mice brain sections were collected 2 days post JEV infection. Enhanced levels of <t>Alexa</t> 488 florescence was observed in JEV infected brain section (Magnification 20x, Scale bar 100 μm). All experiments were performed in triplicates. The data are shown as mean ± S.E from three independent experiments. The fold change is significant where *denotes P
    Alexa 488 Conjugated Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1522 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti rabbit igg1
    ROS is produced in both prion-infected and GDL-exposed COCS. ( A ) COCS were exposed to POM1 for 8 h-10 days, and neuronal loss was assessed by NeuN + pixel coverage. Neuronal loss was significant at 3 dpe. Untreated slices (ut), or slices exposed to pooled <t>IgG,</t> were used as controls (grey). ( B ) In prion-infected COCS (+) neuronal loss became significant at 45 dpi. Controls (-): exposure to non-infectious brain homogenate. Data were analyzed using a two-tailed t-test; n = 9 biological replicates. ( C—D ) COCS were exposed to DHE, and the number of positive fluorescent particles was counted per 10x view field. ( C ) DHE conversion assays were performed at various time points between 1 h and 240 h. Data from three experiments are represented in this graph; n = 27 biological replicates. Slices that were untreated or exposed to pooled IgG were used as controls (grey). ROS production was detectable 4 hours after POM1 exposure and after 1 h with a higher POM1 concentration (335 nM). Here and henceforth data are presented as average ± s.d. and were analyzed by one-way ANOVA with Dunnett’s post-hoc test unless otherwise specified (***: p
    Goat Anti Rabbit Igg1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 278 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher 594 goat anti rabbit igg1 conjugate
    ROS is produced in both prion-infected and GDL-exposed COCS. ( A ) COCS were exposed to POM1 for 8 h-10 days, and neuronal loss was assessed by NeuN + pixel coverage. Neuronal loss was significant at 3 dpe. Untreated slices (ut), or slices exposed to pooled <t>IgG,</t> were used as controls (grey). ( B ) In prion-infected COCS (+) neuronal loss became significant at 45 dpi. Controls (-): exposure to non-infectious brain homogenate. Data were analyzed using a two-tailed t-test; n = 9 biological replicates. ( C—D ) COCS were exposed to DHE, and the number of positive fluorescent particles was counted per 10x view field. ( C ) DHE conversion assays were performed at various time points between 1 h and 240 h. Data from three experiments are represented in this graph; n = 27 biological replicates. Slices that were untreated or exposed to pooled IgG were used as controls (grey). ROS production was detectable 4 hours after POM1 exposure and after 1 h with a higher POM1 concentration (335 nM). Here and henceforth data are presented as average ± s.d. and were analyzed by one-way ANOVA with Dunnett’s post-hoc test unless otherwise specified (***: p
    594 Goat Anti Rabbit Igg1 Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alexa 488 goat anti rabbit igg1
    ROS is produced in both prion-infected and GDL-exposed COCS. ( A ) COCS were exposed to POM1 for 8 h-10 days, and neuronal loss was assessed by NeuN + pixel coverage. Neuronal loss was significant at 3 dpe. Untreated slices (ut), or slices exposed to pooled <t>IgG,</t> were used as controls (grey). ( B ) In prion-infected COCS (+) neuronal loss became significant at 45 dpi. Controls (-): exposure to non-infectious brain homogenate. Data were analyzed using a two-tailed t-test; n = 9 biological replicates. ( C—D ) COCS were exposed to DHE, and the number of positive fluorescent particles was counted per 10x view field. ( C ) DHE conversion assays were performed at various time points between 1 h and 240 h. Data from three experiments are represented in this graph; n = 27 biological replicates. Slices that were untreated or exposed to pooled IgG were used as controls (grey). ROS production was detectable 4 hours after POM1 exposure and after 1 h with a higher POM1 concentration (335 nM). Here and henceforth data are presented as average ± s.d. and were analyzed by one-way ANOVA with Dunnett’s post-hoc test unless otherwise specified (***: p
    Alexa 488 Goat Anti Rabbit Igg1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti rabbit igg1 alexa fluor 488
    ROS is produced in both prion-infected and GDL-exposed COCS. ( A ) COCS were exposed to POM1 for 8 h-10 days, and neuronal loss was assessed by NeuN + pixel coverage. Neuronal loss was significant at 3 dpe. Untreated slices (ut), or slices exposed to pooled <t>IgG,</t> were used as controls (grey). ( B ) In prion-infected COCS (+) neuronal loss became significant at 45 dpi. Controls (-): exposure to non-infectious brain homogenate. Data were analyzed using a two-tailed t-test; n = 9 biological replicates. ( C—D ) COCS were exposed to DHE, and the number of positive fluorescent particles was counted per 10x view field. ( C ) DHE conversion assays were performed at various time points between 1 h and 240 h. Data from three experiments are represented in this graph; n = 27 biological replicates. Slices that were untreated or exposed to pooled IgG were used as controls (grey). ROS production was detectable 4 hours after POM1 exposure and after 1 h with a higher POM1 concentration (335 nM). Here and henceforth data are presented as average ± s.d. and were analyzed by one-way ANOVA with Dunnett’s post-hoc test unless otherwise specified (***: p
    Goat Anti Rabbit Igg1 Alexa Fluor 488, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher 594 goat anti rabbit igg
    Binding of neuromyelitis optica (NMO)–immunoglobulin G <t>(IgG)</t> to human astrocyte cell lines (A, B) Fixed cells were incubated with NMO-IgG (pooled from patient sera), control IgG (pooled from healthy donor sera), and commercial aquaporin-4 (AQP4) IgG (rabbit polyclonal, cytoplasmic epitopes). AlexaFluor 488 goat anti-human IgG was used to probe for bound NMO-IgG and control IgG, and AlexaFluor <t>594</t> goat anti-rabbit IgG to probe for commercial AQP4 IgG. (A: A4 cells; B: A cells). Nuclear counterstaining with TO-PRO-3 demonstrates the presence of cells in B. Scale bar, 10 μm. (C) Exposure of living A4 cells to NMO-IgG, but not to control IgG, leads to clustering/internalization of surface AQP4. Counterstaining with Phalloidin Alexa 488, after fixing and permeabilization, shows filamentous actin. Scale bar, 10 μm.
    594 Goat Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 83 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher 555 goat anti rabbit igg
    Binding of neuromyelitis optica (NMO)–immunoglobulin G <t>(IgG)</t> to human astrocyte cell lines (A, B) Fixed cells were incubated with NMO-IgG (pooled from patient sera), control IgG (pooled from healthy donor sera), and commercial aquaporin-4 (AQP4) IgG (rabbit polyclonal, cytoplasmic epitopes). AlexaFluor 488 goat anti-human IgG was used to probe for bound NMO-IgG and control IgG, and AlexaFluor <t>594</t> goat anti-rabbit IgG to probe for commercial AQP4 IgG. (A: A4 cells; B: A cells). Nuclear counterstaining with TO-PRO-3 demonstrates the presence of cells in B. Scale bar, 10 μm. (C) Exposure of living A4 cells to NMO-IgG, but not to control IgG, leads to clustering/internalization of surface AQP4. Counterstaining with Phalloidin Alexa 488, after fixing and permeabilization, shows filamentous actin. Scale bar, 10 μm.
    555 Goat Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher 488 goat anti rabbit igg
    GIMAP6 expression and protein distribution in T lymphocytes. A , cell lines representing the various different hematopoietic lineages, including T lymphocytes (Jurkat), promyeloblasts (HL-60), monocytes (U-937), lymphoblasts (K-562), and B lymphocytes (Raji and Ramos), as well as cell lines derived from colon (HCT-15), kidney (293T), liver (Huh-7), and lung (A549 and H1299), were analyzed for GIMAP6 protein expression by immunoblot analysis. The amount of protein loaded onto each lane was estimated by anti-β-actin or anti-GAPDH. B , GIMAP6 RNA expression levels in the hematopoietic cell lines were analyzed by quantitative PCR assay. The results were normalized against the expression level of HPRT1. Error bars indicate standard deviation. C , GIMAP6 RNA expression in three subsets of human PBMCs: CD3+/CD4+, CD3+/CD8+, and CD3− cells. These were analyzed by quantitative PCR assay. The PBMCs were obtained from a healthy donor. HPRT1 was used to normalize the dataset. Error bars indicate standard deviation. D and E , cellular distribution of endogenous GIMAP6 in Jurkat T cells. Immunoblot analysis of various protein preparations, including that of the total cell lysate ( Input ), revealed that endogenous GIMAP6 is restricted to the cytosolic fraction of Jurkat T lymphocyte lysates ( D ). E , confocal laser-scanning microscopy showed that GIMAP6 ( purple , Alexa Fluor 647 goat anti-rabbit <t>IgG)</t> co-localized with the cytosolic marker GAPDH ( green , Alexa Fluor <t>488</t> goat anti-mouse IgG). DAPI ( blue ) was used as a nuclear tracking dye. F , cellular distribution of endogenous GIMAP6 in primary CD3+ T cells. Primary CD3+ T cells enriched from PBMCs were used to perform immunofluorescence microscopy, which showed that GIMAP6 ( green , Alexa Fluor 488 goat anti-rabbit IgG) co-localized with the cytosolic marker GAPDH ( red , Alexa Fluor 594 goat anti-mouse IgG). DAPI ( blue ) was used as a nuclear tracking dye.
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    Thermo Fisher alexafluor488 goat anti rabbit igg
    GIMAP6 expression and protein distribution in T lymphocytes. A , cell lines representing the various different hematopoietic lineages, including T lymphocytes (Jurkat), promyeloblasts (HL-60), monocytes (U-937), lymphoblasts (K-562), and B lymphocytes (Raji and Ramos), as well as cell lines derived from colon (HCT-15), kidney (293T), liver (Huh-7), and lung (A549 and H1299), were analyzed for GIMAP6 protein expression by immunoblot analysis. The amount of protein loaded onto each lane was estimated by anti-β-actin or anti-GAPDH. B , GIMAP6 RNA expression levels in the hematopoietic cell lines were analyzed by quantitative PCR assay. The results were normalized against the expression level of HPRT1. Error bars indicate standard deviation. C , GIMAP6 RNA expression in three subsets of human PBMCs: CD3+/CD4+, CD3+/CD8+, and CD3− cells. These were analyzed by quantitative PCR assay. The PBMCs were obtained from a healthy donor. HPRT1 was used to normalize the dataset. Error bars indicate standard deviation. D and E , cellular distribution of endogenous GIMAP6 in Jurkat T cells. Immunoblot analysis of various protein preparations, including that of the total cell lysate ( Input ), revealed that endogenous GIMAP6 is restricted to the cytosolic fraction of Jurkat T lymphocyte lysates ( D ). E , confocal laser-scanning microscopy showed that GIMAP6 ( purple , Alexa Fluor 647 goat anti-rabbit <t>IgG)</t> co-localized with the cytosolic marker GAPDH ( green , Alexa Fluor <t>488</t> goat anti-mouse IgG). DAPI ( blue ) was used as a nuclear tracking dye. F , cellular distribution of endogenous GIMAP6 in primary CD3+ T cells. Primary CD3+ T cells enriched from PBMCs were used to perform immunofluorescence microscopy, which showed that GIMAP6 ( green , Alexa Fluor 488 goat anti-rabbit IgG) co-localized with the cytosolic marker GAPDH ( red , Alexa Fluor 594 goat anti-mouse IgG). DAPI ( blue ) was used as a nuclear tracking dye.
    Alexafluor488 Goat Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alexa486 goat anti rabbit igg
    GIMAP6 expression and protein distribution in T lymphocytes. A , cell lines representing the various different hematopoietic lineages, including T lymphocytes (Jurkat), promyeloblasts (HL-60), monocytes (U-937), lymphoblasts (K-562), and B lymphocytes (Raji and Ramos), as well as cell lines derived from colon (HCT-15), kidney (293T), liver (Huh-7), and lung (A549 and H1299), were analyzed for GIMAP6 protein expression by immunoblot analysis. The amount of protein loaded onto each lane was estimated by anti-β-actin or anti-GAPDH. B , GIMAP6 RNA expression levels in the hematopoietic cell lines were analyzed by quantitative PCR assay. The results were normalized against the expression level of HPRT1. Error bars indicate standard deviation. C , GIMAP6 RNA expression in three subsets of human PBMCs: CD3+/CD4+, CD3+/CD8+, and CD3− cells. These were analyzed by quantitative PCR assay. The PBMCs were obtained from a healthy donor. HPRT1 was used to normalize the dataset. Error bars indicate standard deviation. D and E , cellular distribution of endogenous GIMAP6 in Jurkat T cells. Immunoblot analysis of various protein preparations, including that of the total cell lysate ( Input ), revealed that endogenous GIMAP6 is restricted to the cytosolic fraction of Jurkat T lymphocyte lysates ( D ). E , confocal laser-scanning microscopy showed that GIMAP6 ( purple , Alexa Fluor 647 goat anti-rabbit <t>IgG)</t> co-localized with the cytosolic marker GAPDH ( green , Alexa Fluor <t>488</t> goat anti-mouse IgG). DAPI ( blue ) was used as a nuclear tracking dye. F , cellular distribution of endogenous GIMAP6 in primary CD3+ T cells. Primary CD3+ T cells enriched from PBMCs were used to perform immunofluorescence microscopy, which showed that GIMAP6 ( green , Alexa Fluor 488 goat anti-rabbit IgG) co-localized with the cytosolic marker GAPDH ( red , Alexa Fluor 594 goat anti-mouse IgG). DAPI ( blue ) was used as a nuclear tracking dye.
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    Thermo Fisher alexafluor594 goat anti rabbit igg
    GIMAP6 expression and protein distribution in T lymphocytes. A , cell lines representing the various different hematopoietic lineages, including T lymphocytes (Jurkat), promyeloblasts (HL-60), monocytes (U-937), lymphoblasts (K-562), and B lymphocytes (Raji and Ramos), as well as cell lines derived from colon (HCT-15), kidney (293T), liver (Huh-7), and lung (A549 and H1299), were analyzed for GIMAP6 protein expression by immunoblot analysis. The amount of protein loaded onto each lane was estimated by anti-β-actin or anti-GAPDH. B , GIMAP6 RNA expression levels in the hematopoietic cell lines were analyzed by quantitative PCR assay. The results were normalized against the expression level of HPRT1. Error bars indicate standard deviation. C , GIMAP6 RNA expression in three subsets of human PBMCs: CD3+/CD4+, CD3+/CD8+, and CD3− cells. These were analyzed by quantitative PCR assay. The PBMCs were obtained from a healthy donor. HPRT1 was used to normalize the dataset. Error bars indicate standard deviation. D and E , cellular distribution of endogenous GIMAP6 in Jurkat T cells. Immunoblot analysis of various protein preparations, including that of the total cell lysate ( Input ), revealed that endogenous GIMAP6 is restricted to the cytosolic fraction of Jurkat T lymphocyte lysates ( D ). E , confocal laser-scanning microscopy showed that GIMAP6 ( purple , Alexa Fluor 647 goat anti-rabbit <t>IgG)</t> co-localized with the cytosolic marker GAPDH ( green , Alexa Fluor <t>488</t> goat anti-mouse IgG). DAPI ( blue ) was used as a nuclear tracking dye. F , cellular distribution of endogenous GIMAP6 in primary CD3+ T cells. Primary CD3+ T cells enriched from PBMCs were used to perform immunofluorescence microscopy, which showed that GIMAP6 ( green , Alexa Fluor 488 goat anti-rabbit IgG) co-localized with the cytosolic marker GAPDH ( red , Alexa Fluor 594 goat anti-mouse IgG). DAPI ( blue ) was used as a nuclear tracking dye.
    Alexafluor594 Goat Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti rabbit igg 680
    GIMAP6 expression and protein distribution in T lymphocytes. A , cell lines representing the various different hematopoietic lineages, including T lymphocytes (Jurkat), promyeloblasts (HL-60), monocytes (U-937), lymphoblasts (K-562), and B lymphocytes (Raji and Ramos), as well as cell lines derived from colon (HCT-15), kidney (293T), liver (Huh-7), and lung (A549 and H1299), were analyzed for GIMAP6 protein expression by immunoblot analysis. The amount of protein loaded onto each lane was estimated by anti-β-actin or anti-GAPDH. B , GIMAP6 RNA expression levels in the hematopoietic cell lines were analyzed by quantitative PCR assay. The results were normalized against the expression level of HPRT1. Error bars indicate standard deviation. C , GIMAP6 RNA expression in three subsets of human PBMCs: CD3+/CD4+, CD3+/CD8+, and CD3− cells. These were analyzed by quantitative PCR assay. The PBMCs were obtained from a healthy donor. HPRT1 was used to normalize the dataset. Error bars indicate standard deviation. D and E , cellular distribution of endogenous GIMAP6 in Jurkat T cells. Immunoblot analysis of various protein preparations, including that of the total cell lysate ( Input ), revealed that endogenous GIMAP6 is restricted to the cytosolic fraction of Jurkat T lymphocyte lysates ( D ). E , confocal laser-scanning microscopy showed that GIMAP6 ( purple , Alexa Fluor 647 goat anti-rabbit <t>IgG)</t> co-localized with the cytosolic marker GAPDH ( green , Alexa Fluor <t>488</t> goat anti-mouse IgG). DAPI ( blue ) was used as a nuclear tracking dye. F , cellular distribution of endogenous GIMAP6 in primary CD3+ T cells. Primary CD3+ T cells enriched from PBMCs were used to perform immunofluorescence microscopy, which showed that GIMAP6 ( green , Alexa Fluor 488 goat anti-rabbit IgG) co-localized with the cytosolic marker GAPDH ( red , Alexa Fluor 594 goat anti-mouse IgG). DAPI ( blue ) was used as a nuclear tracking dye.
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    Thermo Fisher goat anti rabbit igg alexa568
    GIMAP6 expression and protein distribution in T lymphocytes. A , cell lines representing the various different hematopoietic lineages, including T lymphocytes (Jurkat), promyeloblasts (HL-60), monocytes (U-937), lymphoblasts (K-562), and B lymphocytes (Raji and Ramos), as well as cell lines derived from colon (HCT-15), kidney (293T), liver (Huh-7), and lung (A549 and H1299), were analyzed for GIMAP6 protein expression by immunoblot analysis. The amount of protein loaded onto each lane was estimated by anti-β-actin or anti-GAPDH. B , GIMAP6 RNA expression levels in the hematopoietic cell lines were analyzed by quantitative PCR assay. The results were normalized against the expression level of HPRT1. Error bars indicate standard deviation. C , GIMAP6 RNA expression in three subsets of human PBMCs: CD3+/CD4+, CD3+/CD8+, and CD3− cells. These were analyzed by quantitative PCR assay. The PBMCs were obtained from a healthy donor. HPRT1 was used to normalize the dataset. Error bars indicate standard deviation. D and E , cellular distribution of endogenous GIMAP6 in Jurkat T cells. Immunoblot analysis of various protein preparations, including that of the total cell lysate ( Input ), revealed that endogenous GIMAP6 is restricted to the cytosolic fraction of Jurkat T lymphocyte lysates ( D ). E , confocal laser-scanning microscopy showed that GIMAP6 ( purple , Alexa Fluor 647 goat anti-rabbit <t>IgG)</t> co-localized with the cytosolic marker GAPDH ( green , Alexa Fluor <t>488</t> goat anti-mouse IgG). DAPI ( blue ) was used as a nuclear tracking dye. F , cellular distribution of endogenous GIMAP6 in primary CD3+ T cells. Primary CD3+ T cells enriched from PBMCs were used to perform immunofluorescence microscopy, which showed that GIMAP6 ( green , Alexa Fluor 488 goat anti-rabbit IgG) co-localized with the cytosolic marker GAPDH ( red , Alexa Fluor 594 goat anti-mouse IgG). DAPI ( blue ) was used as a nuclear tracking dye.
    Goat Anti Rabbit Igg Alexa568, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Thermo Fisher alexafluor647 goat anti rabbit igg
    GIMAP6 expression and protein distribution in T lymphocytes. A , cell lines representing the various different hematopoietic lineages, including T lymphocytes (Jurkat), promyeloblasts (HL-60), monocytes (U-937), lymphoblasts (K-562), and B lymphocytes (Raji and Ramos), as well as cell lines derived from colon (HCT-15), kidney (293T), liver (Huh-7), and lung (A549 and H1299), were analyzed for GIMAP6 protein expression by immunoblot analysis. The amount of protein loaded onto each lane was estimated by anti-β-actin or anti-GAPDH. B , GIMAP6 RNA expression levels in the hematopoietic cell lines were analyzed by quantitative PCR assay. The results were normalized against the expression level of HPRT1. Error bars indicate standard deviation. C , GIMAP6 RNA expression in three subsets of human PBMCs: CD3+/CD4+, CD3+/CD8+, and CD3− cells. These were analyzed by quantitative PCR assay. The PBMCs were obtained from a healthy donor. HPRT1 was used to normalize the dataset. Error bars indicate standard deviation. D and E , cellular distribution of endogenous GIMAP6 in Jurkat T cells. Immunoblot analysis of various protein preparations, including that of the total cell lysate ( Input ), revealed that endogenous GIMAP6 is restricted to the cytosolic fraction of Jurkat T lymphocyte lysates ( D ). E , confocal laser-scanning microscopy showed that GIMAP6 ( purple , Alexa Fluor 647 goat anti-rabbit <t>IgG)</t> co-localized with the cytosolic marker GAPDH ( green , Alexa Fluor <t>488</t> goat anti-mouse IgG). DAPI ( blue ) was used as a nuclear tracking dye. F , cellular distribution of endogenous GIMAP6 in primary CD3+ T cells. Primary CD3+ T cells enriched from PBMCs were used to perform immunofluorescence microscopy, which showed that GIMAP6 ( green , Alexa Fluor 488 goat anti-rabbit IgG) co-localized with the cytosolic marker GAPDH ( red , Alexa Fluor 594 goat anti-mouse IgG). DAPI ( blue ) was used as a nuclear tracking dye.
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    Thermo Fisher goat anti rabbit igg af488
    SLO injury of the PM reduces BCR activation and internalization. (A–D) BCR–CTB coclustering in the presence or absence of SLO. B cells were incubated at 4°C with Cy3-Fab (5 µg/ml; −XL) or AF546-F(ab′) 2 anti–mouse <t>IgM+IgG</t> (5 µg/ml; +XL) plus <t>AF488-CTB</t> (3 µg/ml) and warmed to 37°C for 5 min with or without SLO, followed by fixation and confocal microscopy. (A) Representative images. Bar, 2.5 µm. (B) Percentages (± SD) of all B cells exhibiting polarized BCR clusters (capping). (C) Percentages of B cells with polarized BCR clusters showing impaired BCR caps. (D) Percentages of B cells with polarized BCR clusters showing BCR–CTB coclustering. Data in B–D were generated by visual inspection of images from four independent experiments. (E and F) Tyrosine phosphorylation (pY) of B cells treated with or without SLO. B cells were incubated at 4°C with SLO and AF546-F(ab′) 2 -goat anti–mouse IgM+IgG (5 µg/ml) and warmed to 37°C for 5 min, followed by fixation, permeabilization, and staining for phosphotyrosine (pY) and analysis of confocal microscopy (E) and flow cytometry (F). Shown are representative images and the mean MFI (± SD) of pY from three independent experiments. Bar, 2.5 µm. (G) Effect of SLO treatment on BCR internalization. B cells were incubated at 4°C with biotinylated F(ab′) 2 anti–mouse IgM+IgG (10 µg/ml), followed by 37°C incubation with or without SLO for the indicated times. Cells were then labeled with PE-streptavidin at 4°C and analyzed by flow cytometry to determine the percentage of surface-labeled BCRs remaining on the cell surface. Shown is the mean percentage (± SD) from three independent experiments. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P
    Goat Anti Rabbit Igg Af488, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 151 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti rabbit igg af568
    SLO injury of the PM reduces BCR activation and internalization. (A–D) BCR–CTB coclustering in the presence or absence of SLO. B cells were incubated at 4°C with Cy3-Fab (5 µg/ml; −XL) or AF546-F(ab′) 2 anti–mouse <t>IgM+IgG</t> (5 µg/ml; +XL) plus <t>AF488-CTB</t> (3 µg/ml) and warmed to 37°C for 5 min with or without SLO, followed by fixation and confocal microscopy. (A) Representative images. Bar, 2.5 µm. (B) Percentages (± SD) of all B cells exhibiting polarized BCR clusters (capping). (C) Percentages of B cells with polarized BCR clusters showing impaired BCR caps. (D) Percentages of B cells with polarized BCR clusters showing BCR–CTB coclustering. Data in B–D were generated by visual inspection of images from four independent experiments. (E and F) Tyrosine phosphorylation (pY) of B cells treated with or without SLO. B cells were incubated at 4°C with SLO and AF546-F(ab′) 2 -goat anti–mouse IgM+IgG (5 µg/ml) and warmed to 37°C for 5 min, followed by fixation, permeabilization, and staining for phosphotyrosine (pY) and analysis of confocal microscopy (E) and flow cytometry (F). Shown are representative images and the mean MFI (± SD) of pY from three independent experiments. Bar, 2.5 µm. (G) Effect of SLO treatment on BCR internalization. B cells were incubated at 4°C with biotinylated F(ab′) 2 anti–mouse IgM+IgG (10 µg/ml), followed by 37°C incubation with or without SLO for the indicated times. Cells were then labeled with PE-streptavidin at 4°C and analyzed by flow cytometry to determine the percentage of surface-labeled BCRs remaining on the cell surface. Shown is the mean percentage (± SD) from three independent experiments. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P
    Goat Anti Rabbit Igg Af568, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher cy3 goat anti rabbit igg
    SLO injury of the PM reduces BCR activation and internalization. (A–D) BCR–CTB coclustering in the presence or absence of SLO. B cells were incubated at 4°C with Cy3-Fab (5 µg/ml; −XL) or AF546-F(ab′) 2 anti–mouse <t>IgM+IgG</t> (5 µg/ml; +XL) plus <t>AF488-CTB</t> (3 µg/ml) and warmed to 37°C for 5 min with or without SLO, followed by fixation and confocal microscopy. (A) Representative images. Bar, 2.5 µm. (B) Percentages (± SD) of all B cells exhibiting polarized BCR clusters (capping). (C) Percentages of B cells with polarized BCR clusters showing impaired BCR caps. (D) Percentages of B cells with polarized BCR clusters showing BCR–CTB coclustering. Data in B–D were generated by visual inspection of images from four independent experiments. (E and F) Tyrosine phosphorylation (pY) of B cells treated with or without SLO. B cells were incubated at 4°C with SLO and AF546-F(ab′) 2 -goat anti–mouse IgM+IgG (5 µg/ml) and warmed to 37°C for 5 min, followed by fixation, permeabilization, and staining for phosphotyrosine (pY) and analysis of confocal microscopy (E) and flow cytometry (F). Shown are representative images and the mean MFI (± SD) of pY from three independent experiments. Bar, 2.5 µm. (G) Effect of SLO treatment on BCR internalization. B cells were incubated at 4°C with biotinylated F(ab′) 2 anti–mouse IgM+IgG (10 µg/ml), followed by 37°C incubation with or without SLO for the indicated times. Cells were then labeled with PE-streptavidin at 4°C and analyzed by flow cytometry to determine the percentage of surface-labeled BCRs remaining on the cell surface. Shown is the mean percentage (± SD) from three independent experiments. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P
    Cy3 Goat Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 77 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alexa594 goat anti rabbit igg
    SLO injury of the PM reduces BCR activation and internalization. (A–D) BCR–CTB coclustering in the presence or absence of SLO. B cells were incubated at 4°C with Cy3-Fab (5 µg/ml; −XL) or AF546-F(ab′) 2 anti–mouse <t>IgM+IgG</t> (5 µg/ml; +XL) plus <t>AF488-CTB</t> (3 µg/ml) and warmed to 37°C for 5 min with or without SLO, followed by fixation and confocal microscopy. (A) Representative images. Bar, 2.5 µm. (B) Percentages (± SD) of all B cells exhibiting polarized BCR clusters (capping). (C) Percentages of B cells with polarized BCR clusters showing impaired BCR caps. (D) Percentages of B cells with polarized BCR clusters showing BCR–CTB coclustering. Data in B–D were generated by visual inspection of images from four independent experiments. (E and F) Tyrosine phosphorylation (pY) of B cells treated with or without SLO. B cells were incubated at 4°C with SLO and AF546-F(ab′) 2 -goat anti–mouse IgM+IgG (5 µg/ml) and warmed to 37°C for 5 min, followed by fixation, permeabilization, and staining for phosphotyrosine (pY) and analysis of confocal microscopy (E) and flow cytometry (F). Shown are representative images and the mean MFI (± SD) of pY from three independent experiments. Bar, 2.5 µm. (G) Effect of SLO treatment on BCR internalization. B cells were incubated at 4°C with biotinylated F(ab′) 2 anti–mouse IgM+IgG (10 µg/ml), followed by 37°C incubation with or without SLO for the indicated times. Cells were then labeled with PE-streptavidin at 4°C and analyzed by flow cytometry to determine the percentage of surface-labeled BCRs remaining on the cell surface. Shown is the mean percentage (± SD) from three independent experiments. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P
    Alexa594 Goat Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti rabbit igg alexa
    SLO injury of the PM reduces BCR activation and internalization. (A–D) BCR–CTB coclustering in the presence or absence of SLO. B cells were incubated at 4°C with Cy3-Fab (5 µg/ml; −XL) or AF546-F(ab′) 2 anti–mouse <t>IgM+IgG</t> (5 µg/ml; +XL) plus <t>AF488-CTB</t> (3 µg/ml) and warmed to 37°C for 5 min with or without SLO, followed by fixation and confocal microscopy. (A) Representative images. Bar, 2.5 µm. (B) Percentages (± SD) of all B cells exhibiting polarized BCR clusters (capping). (C) Percentages of B cells with polarized BCR clusters showing impaired BCR caps. (D) Percentages of B cells with polarized BCR clusters showing BCR–CTB coclustering. Data in B–D were generated by visual inspection of images from four independent experiments. (E and F) Tyrosine phosphorylation (pY) of B cells treated with or without SLO. B cells were incubated at 4°C with SLO and AF546-F(ab′) 2 -goat anti–mouse IgM+IgG (5 µg/ml) and warmed to 37°C for 5 min, followed by fixation, permeabilization, and staining for phosphotyrosine (pY) and analysis of confocal microscopy (E) and flow cytometry (F). Shown are representative images and the mean MFI (± SD) of pY from three independent experiments. Bar, 2.5 µm. (G) Effect of SLO treatment on BCR internalization. B cells were incubated at 4°C with biotinylated F(ab′) 2 anti–mouse IgM+IgG (10 µg/ml), followed by 37°C incubation with or without SLO for the indicated times. Cells were then labeled with PE-streptavidin at 4°C and analyzed by flow cytometry to determine the percentage of surface-labeled BCRs remaining on the cell surface. Shown is the mean percentage (± SD) from three independent experiments. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P
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    Thermo Fisher goat anti rabbit igg 647
    SLO injury of the PM reduces BCR activation and internalization. (A–D) BCR–CTB coclustering in the presence or absence of SLO. B cells were incubated at 4°C with Cy3-Fab (5 µg/ml; −XL) or AF546-F(ab′) 2 anti–mouse <t>IgM+IgG</t> (5 µg/ml; +XL) plus <t>AF488-CTB</t> (3 µg/ml) and warmed to 37°C for 5 min with or without SLO, followed by fixation and confocal microscopy. (A) Representative images. Bar, 2.5 µm. (B) Percentages (± SD) of all B cells exhibiting polarized BCR clusters (capping). (C) Percentages of B cells with polarized BCR clusters showing impaired BCR caps. (D) Percentages of B cells with polarized BCR clusters showing BCR–CTB coclustering. Data in B–D were generated by visual inspection of images from four independent experiments. (E and F) Tyrosine phosphorylation (pY) of B cells treated with or without SLO. B cells were incubated at 4°C with SLO and AF546-F(ab′) 2 -goat anti–mouse IgM+IgG (5 µg/ml) and warmed to 37°C for 5 min, followed by fixation, permeabilization, and staining for phosphotyrosine (pY) and analysis of confocal microscopy (E) and flow cytometry (F). Shown are representative images and the mean MFI (± SD) of pY from three independent experiments. Bar, 2.5 µm. (G) Effect of SLO treatment on BCR internalization. B cells were incubated at 4°C with biotinylated F(ab′) 2 anti–mouse IgM+IgG (10 µg/ml), followed by 37°C incubation with or without SLO for the indicated times. Cells were then labeled with PE-streptavidin at 4°C and analyzed by flow cytometry to determine the percentage of surface-labeled BCRs remaining on the cell surface. Shown is the mean percentage (± SD) from three independent experiments. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P
    Goat Anti Rabbit Igg 647, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 81/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alexared goat anti rabbit igg
    SLO injury of the PM reduces BCR activation and internalization. (A–D) BCR–CTB coclustering in the presence or absence of SLO. B cells were incubated at 4°C with Cy3-Fab (5 µg/ml; −XL) or AF546-F(ab′) 2 anti–mouse <t>IgM+IgG</t> (5 µg/ml; +XL) plus <t>AF488-CTB</t> (3 µg/ml) and warmed to 37°C for 5 min with or without SLO, followed by fixation and confocal microscopy. (A) Representative images. Bar, 2.5 µm. (B) Percentages (± SD) of all B cells exhibiting polarized BCR clusters (capping). (C) Percentages of B cells with polarized BCR clusters showing impaired BCR caps. (D) Percentages of B cells with polarized BCR clusters showing BCR–CTB coclustering. Data in B–D were generated by visual inspection of images from four independent experiments. (E and F) Tyrosine phosphorylation (pY) of B cells treated with or without SLO. B cells were incubated at 4°C with SLO and AF546-F(ab′) 2 -goat anti–mouse IgM+IgG (5 µg/ml) and warmed to 37°C for 5 min, followed by fixation, permeabilization, and staining for phosphotyrosine (pY) and analysis of confocal microscopy (E) and flow cytometry (F). Shown are representative images and the mean MFI (± SD) of pY from three independent experiments. Bar, 2.5 µm. (G) Effect of SLO treatment on BCR internalization. B cells were incubated at 4°C with biotinylated F(ab′) 2 anti–mouse IgM+IgG (10 µg/ml), followed by 37°C incubation with or without SLO for the indicated times. Cells were then labeled with PE-streptavidin at 4°C and analyzed by flow cytometry to determine the percentage of surface-labeled BCRs remaining on the cell surface. Shown is the mean percentage (± SD) from three independent experiments. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P
    Alexared Goat Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 75/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti rabbit igg alexafluor568
    SLO injury of the PM reduces BCR activation and internalization. (A–D) BCR–CTB coclustering in the presence or absence of SLO. B cells were incubated at 4°C with Cy3-Fab (5 µg/ml; −XL) or AF546-F(ab′) 2 anti–mouse <t>IgM+IgG</t> (5 µg/ml; +XL) plus <t>AF488-CTB</t> (3 µg/ml) and warmed to 37°C for 5 min with or without SLO, followed by fixation and confocal microscopy. (A) Representative images. Bar, 2.5 µm. (B) Percentages (± SD) of all B cells exhibiting polarized BCR clusters (capping). (C) Percentages of B cells with polarized BCR clusters showing impaired BCR caps. (D) Percentages of B cells with polarized BCR clusters showing BCR–CTB coclustering. Data in B–D were generated by visual inspection of images from four independent experiments. (E and F) Tyrosine phosphorylation (pY) of B cells treated with or without SLO. B cells were incubated at 4°C with SLO and AF546-F(ab′) 2 -goat anti–mouse IgM+IgG (5 µg/ml) and warmed to 37°C for 5 min, followed by fixation, permeabilization, and staining for phosphotyrosine (pY) and analysis of confocal microscopy (E) and flow cytometry (F). Shown are representative images and the mean MFI (± SD) of pY from three independent experiments. Bar, 2.5 µm. (G) Effect of SLO treatment on BCR internalization. B cells were incubated at 4°C with biotinylated F(ab′) 2 anti–mouse IgM+IgG (10 µg/ml), followed by 37°C incubation with or without SLO for the indicated times. Cells were then labeled with PE-streptavidin at 4°C and analyzed by flow cytometry to determine the percentage of surface-labeled BCRs remaining on the cell surface. Shown is the mean percentage (± SD) from three independent experiments. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P
    Goat Anti Rabbit Igg Alexafluor568, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 74 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti rabbit igg alexa546
    SLO injury of the PM reduces BCR activation and internalization. (A–D) BCR–CTB coclustering in the presence or absence of SLO. B cells were incubated at 4°C with Cy3-Fab (5 µg/ml; −XL) or AF546-F(ab′) 2 anti–mouse <t>IgM+IgG</t> (5 µg/ml; +XL) plus <t>AF488-CTB</t> (3 µg/ml) and warmed to 37°C for 5 min with or without SLO, followed by fixation and confocal microscopy. (A) Representative images. Bar, 2.5 µm. (B) Percentages (± SD) of all B cells exhibiting polarized BCR clusters (capping). (C) Percentages of B cells with polarized BCR clusters showing impaired BCR caps. (D) Percentages of B cells with polarized BCR clusters showing BCR–CTB coclustering. Data in B–D were generated by visual inspection of images from four independent experiments. (E and F) Tyrosine phosphorylation (pY) of B cells treated with or without SLO. B cells were incubated at 4°C with SLO and AF546-F(ab′) 2 -goat anti–mouse IgM+IgG (5 µg/ml) and warmed to 37°C for 5 min, followed by fixation, permeabilization, and staining for phosphotyrosine (pY) and analysis of confocal microscopy (E) and flow cytometry (F). Shown are representative images and the mean MFI (± SD) of pY from three independent experiments. Bar, 2.5 µm. (G) Effect of SLO treatment on BCR internalization. B cells were incubated at 4°C with biotinylated F(ab′) 2 anti–mouse IgM+IgG (10 µg/ml), followed by 37°C incubation with or without SLO for the indicated times. Cells were then labeled with PE-streptavidin at 4°C and analyzed by flow cytometry to determine the percentage of surface-labeled BCRs remaining on the cell surface. Shown is the mean percentage (± SD) from three independent experiments. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P
    Goat Anti Rabbit Igg Alexa546, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alexafluor546 goat anti rabbit igg
    SLO injury of the PM reduces BCR activation and internalization. (A–D) BCR–CTB coclustering in the presence or absence of SLO. B cells were incubated at 4°C with Cy3-Fab (5 µg/ml; −XL) or AF546-F(ab′) 2 anti–mouse <t>IgM+IgG</t> (5 µg/ml; +XL) plus <t>AF488-CTB</t> (3 µg/ml) and warmed to 37°C for 5 min with or without SLO, followed by fixation and confocal microscopy. (A) Representative images. Bar, 2.5 µm. (B) Percentages (± SD) of all B cells exhibiting polarized BCR clusters (capping). (C) Percentages of B cells with polarized BCR clusters showing impaired BCR caps. (D) Percentages of B cells with polarized BCR clusters showing BCR–CTB coclustering. Data in B–D were generated by visual inspection of images from four independent experiments. (E and F) Tyrosine phosphorylation (pY) of B cells treated with or without SLO. B cells were incubated at 4°C with SLO and AF546-F(ab′) 2 -goat anti–mouse IgM+IgG (5 µg/ml) and warmed to 37°C for 5 min, followed by fixation, permeabilization, and staining for phosphotyrosine (pY) and analysis of confocal microscopy (E) and flow cytometry (F). Shown are representative images and the mean MFI (± SD) of pY from three independent experiments. Bar, 2.5 µm. (G) Effect of SLO treatment on BCR internalization. B cells were incubated at 4°C with biotinylated F(ab′) 2 anti–mouse IgM+IgG (10 µg/ml), followed by 37°C incubation with or without SLO for the indicated times. Cells were then labeled with PE-streptavidin at 4°C and analyzed by flow cytometry to determine the percentage of surface-labeled BCRs remaining on the cell surface. Shown is the mean percentage (± SD) from three independent experiments. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P
    Alexafluor546 Goat Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher fluorescent goat anti rabbit igg
    SLO injury of the PM reduces BCR activation and internalization. (A–D) BCR–CTB coclustering in the presence or absence of SLO. B cells were incubated at 4°C with Cy3-Fab (5 µg/ml; −XL) or AF546-F(ab′) 2 anti–mouse <t>IgM+IgG</t> (5 µg/ml; +XL) plus <t>AF488-CTB</t> (3 µg/ml) and warmed to 37°C for 5 min with or without SLO, followed by fixation and confocal microscopy. (A) Representative images. Bar, 2.5 µm. (B) Percentages (± SD) of all B cells exhibiting polarized BCR clusters (capping). (C) Percentages of B cells with polarized BCR clusters showing impaired BCR caps. (D) Percentages of B cells with polarized BCR clusters showing BCR–CTB coclustering. Data in B–D were generated by visual inspection of images from four independent experiments. (E and F) Tyrosine phosphorylation (pY) of B cells treated with or without SLO. B cells were incubated at 4°C with SLO and AF546-F(ab′) 2 -goat anti–mouse IgM+IgG (5 µg/ml) and warmed to 37°C for 5 min, followed by fixation, permeabilization, and staining for phosphotyrosine (pY) and analysis of confocal microscopy (E) and flow cytometry (F). Shown are representative images and the mean MFI (± SD) of pY from three independent experiments. Bar, 2.5 µm. (G) Effect of SLO treatment on BCR internalization. B cells were incubated at 4°C with biotinylated F(ab′) 2 anti–mouse IgM+IgG (10 µg/ml), followed by 37°C incubation with or without SLO for the indicated times. Cells were then labeled with PE-streptavidin at 4°C and analyzed by flow cytometry to determine the percentage of surface-labeled BCRs remaining on the cell surface. Shown is the mean percentage (± SD) from three independent experiments. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P
    fluorescent Goat Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 87/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher 800 goat anti rabbit igg
    SLO injury of the PM reduces BCR activation and internalization. (A–D) BCR–CTB coclustering in the presence or absence of SLO. B cells were incubated at 4°C with Cy3-Fab (5 µg/ml; −XL) or AF546-F(ab′) 2 anti–mouse <t>IgM+IgG</t> (5 µg/ml; +XL) plus <t>AF488-CTB</t> (3 µg/ml) and warmed to 37°C for 5 min with or without SLO, followed by fixation and confocal microscopy. (A) Representative images. Bar, 2.5 µm. (B) Percentages (± SD) of all B cells exhibiting polarized BCR clusters (capping). (C) Percentages of B cells with polarized BCR clusters showing impaired BCR caps. (D) Percentages of B cells with polarized BCR clusters showing BCR–CTB coclustering. Data in B–D were generated by visual inspection of images from four independent experiments. (E and F) Tyrosine phosphorylation (pY) of B cells treated with or without SLO. B cells were incubated at 4°C with SLO and AF546-F(ab′) 2 -goat anti–mouse IgM+IgG (5 µg/ml) and warmed to 37°C for 5 min, followed by fixation, permeabilization, and staining for phosphotyrosine (pY) and analysis of confocal microscopy (E) and flow cytometry (F). Shown are representative images and the mean MFI (± SD) of pY from three independent experiments. Bar, 2.5 µm. (G) Effect of SLO treatment on BCR internalization. B cells were incubated at 4°C with biotinylated F(ab′) 2 anti–mouse IgM+IgG (10 µg/ml), followed by 37°C incubation with or without SLO for the indicated times. Cells were then labeled with PE-streptavidin at 4°C and analyzed by flow cytometry to determine the percentage of surface-labeled BCRs remaining on the cell surface. Shown is the mean percentage (± SD) from three independent experiments. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P
    800 Goat Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti rabbit igg a488
    SLO injury of the PM reduces BCR activation and internalization. (A–D) BCR–CTB coclustering in the presence or absence of SLO. B cells were incubated at 4°C with Cy3-Fab (5 µg/ml; −XL) or AF546-F(ab′) 2 anti–mouse <t>IgM+IgG</t> (5 µg/ml; +XL) plus <t>AF488-CTB</t> (3 µg/ml) and warmed to 37°C for 5 min with or without SLO, followed by fixation and confocal microscopy. (A) Representative images. Bar, 2.5 µm. (B) Percentages (± SD) of all B cells exhibiting polarized BCR clusters (capping). (C) Percentages of B cells with polarized BCR clusters showing impaired BCR caps. (D) Percentages of B cells with polarized BCR clusters showing BCR–CTB coclustering. Data in B–D were generated by visual inspection of images from four independent experiments. (E and F) Tyrosine phosphorylation (pY) of B cells treated with or without SLO. B cells were incubated at 4°C with SLO and AF546-F(ab′) 2 -goat anti–mouse IgM+IgG (5 µg/ml) and warmed to 37°C for 5 min, followed by fixation, permeabilization, and staining for phosphotyrosine (pY) and analysis of confocal microscopy (E) and flow cytometry (F). Shown are representative images and the mean MFI (± SD) of pY from three independent experiments. Bar, 2.5 µm. (G) Effect of SLO treatment on BCR internalization. B cells were incubated at 4°C with biotinylated F(ab′) 2 anti–mouse IgM+IgG (10 µg/ml), followed by 37°C incubation with or without SLO for the indicated times. Cells were then labeled with PE-streptavidin at 4°C and analyzed by flow cytometry to determine the percentage of surface-labeled BCRs remaining on the cell surface. Shown is the mean percentage (± SD) from three independent experiments. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P
    Goat Anti Rabbit Igg A488, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti rabbit dylight594 igg
    SLO injury of the PM reduces BCR activation and internalization. (A–D) BCR–CTB coclustering in the presence or absence of SLO. B cells were incubated at 4°C with Cy3-Fab (5 µg/ml; −XL) or AF546-F(ab′) 2 anti–mouse <t>IgM+IgG</t> (5 µg/ml; +XL) plus <t>AF488-CTB</t> (3 µg/ml) and warmed to 37°C for 5 min with or without SLO, followed by fixation and confocal microscopy. (A) Representative images. Bar, 2.5 µm. (B) Percentages (± SD) of all B cells exhibiting polarized BCR clusters (capping). (C) Percentages of B cells with polarized BCR clusters showing impaired BCR caps. (D) Percentages of B cells with polarized BCR clusters showing BCR–CTB coclustering. Data in B–D were generated by visual inspection of images from four independent experiments. (E and F) Tyrosine phosphorylation (pY) of B cells treated with or without SLO. B cells were incubated at 4°C with SLO and AF546-F(ab′) 2 -goat anti–mouse IgM+IgG (5 µg/ml) and warmed to 37°C for 5 min, followed by fixation, permeabilization, and staining for phosphotyrosine (pY) and analysis of confocal microscopy (E) and flow cytometry (F). Shown are representative images and the mean MFI (± SD) of pY from three independent experiments. Bar, 2.5 µm. (G) Effect of SLO treatment on BCR internalization. B cells were incubated at 4°C with biotinylated F(ab′) 2 anti–mouse IgM+IgG (10 µg/ml), followed by 37°C incubation with or without SLO for the indicated times. Cells were then labeled with PE-streptavidin at 4°C and analyzed by flow cytometry to determine the percentage of surface-labeled BCRs remaining on the cell surface. Shown is the mean percentage (± SD) from three independent experiments. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P
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    Thermo Fisher magnabind goat anti rabbit igg
    SLO injury of the PM reduces BCR activation and internalization. (A–D) BCR–CTB coclustering in the presence or absence of SLO. B cells were incubated at 4°C with Cy3-Fab (5 µg/ml; −XL) or AF546-F(ab′) 2 anti–mouse <t>IgM+IgG</t> (5 µg/ml; +XL) plus <t>AF488-CTB</t> (3 µg/ml) and warmed to 37°C for 5 min with or without SLO, followed by fixation and confocal microscopy. (A) Representative images. Bar, 2.5 µm. (B) Percentages (± SD) of all B cells exhibiting polarized BCR clusters (capping). (C) Percentages of B cells with polarized BCR clusters showing impaired BCR caps. (D) Percentages of B cells with polarized BCR clusters showing BCR–CTB coclustering. Data in B–D were generated by visual inspection of images from four independent experiments. (E and F) Tyrosine phosphorylation (pY) of B cells treated with or without SLO. B cells were incubated at 4°C with SLO and AF546-F(ab′) 2 -goat anti–mouse IgM+IgG (5 µg/ml) and warmed to 37°C for 5 min, followed by fixation, permeabilization, and staining for phosphotyrosine (pY) and analysis of confocal microscopy (E) and flow cytometry (F). Shown are representative images and the mean MFI (± SD) of pY from three independent experiments. Bar, 2.5 µm. (G) Effect of SLO treatment on BCR internalization. B cells were incubated at 4°C with biotinylated F(ab′) 2 anti–mouse IgM+IgG (10 µg/ml), followed by 37°C incubation with or without SLO for the indicated times. Cells were then labeled with PE-streptavidin at 4°C and analyzed by flow cytometry to determine the percentage of surface-labeled BCRs remaining on the cell surface. Shown is the mean percentage (± SD) from three independent experiments. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P
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    Thermo Fisher alexa514 goat anti rabbit igg
    SLO injury of the PM reduces BCR activation and internalization. (A–D) BCR–CTB coclustering in the presence or absence of SLO. B cells were incubated at 4°C with Cy3-Fab (5 µg/ml; −XL) or AF546-F(ab′) 2 anti–mouse <t>IgM+IgG</t> (5 µg/ml; +XL) plus <t>AF488-CTB</t> (3 µg/ml) and warmed to 37°C for 5 min with or without SLO, followed by fixation and confocal microscopy. (A) Representative images. Bar, 2.5 µm. (B) Percentages (± SD) of all B cells exhibiting polarized BCR clusters (capping). (C) Percentages of B cells with polarized BCR clusters showing impaired BCR caps. (D) Percentages of B cells with polarized BCR clusters showing BCR–CTB coclustering. Data in B–D were generated by visual inspection of images from four independent experiments. (E and F) Tyrosine phosphorylation (pY) of B cells treated with or without SLO. B cells were incubated at 4°C with SLO and AF546-F(ab′) 2 -goat anti–mouse IgM+IgG (5 µg/ml) and warmed to 37°C for 5 min, followed by fixation, permeabilization, and staining for phosphotyrosine (pY) and analysis of confocal microscopy (E) and flow cytometry (F). Shown are representative images and the mean MFI (± SD) of pY from three independent experiments. Bar, 2.5 µm. (G) Effect of SLO treatment on BCR internalization. B cells were incubated at 4°C with biotinylated F(ab′) 2 anti–mouse IgM+IgG (10 µg/ml), followed by 37°C incubation with or without SLO for the indicated times. Cells were then labeled with PE-streptavidin at 4°C and analyzed by flow cytometry to determine the percentage of surface-labeled BCRs remaining on the cell surface. Shown is the mean percentage (± SD) from three independent experiments. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P
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    Thermo Fisher 568 goat anti rabbit igg
    SLO injury of the PM reduces BCR activation and internalization. (A–D) BCR–CTB coclustering in the presence or absence of SLO. B cells were incubated at 4°C with Cy3-Fab (5 µg/ml; −XL) or AF546-F(ab′) 2 anti–mouse <t>IgM+IgG</t> (5 µg/ml; +XL) plus <t>AF488-CTB</t> (3 µg/ml) and warmed to 37°C for 5 min with or without SLO, followed by fixation and confocal microscopy. (A) Representative images. Bar, 2.5 µm. (B) Percentages (± SD) of all B cells exhibiting polarized BCR clusters (capping). (C) Percentages of B cells with polarized BCR clusters showing impaired BCR caps. (D) Percentages of B cells with polarized BCR clusters showing BCR–CTB coclustering. Data in B–D were generated by visual inspection of images from four independent experiments. (E and F) Tyrosine phosphorylation (pY) of B cells treated with or without SLO. B cells were incubated at 4°C with SLO and AF546-F(ab′) 2 -goat anti–mouse IgM+IgG (5 µg/ml) and warmed to 37°C for 5 min, followed by fixation, permeabilization, and staining for phosphotyrosine (pY) and analysis of confocal microscopy (E) and flow cytometry (F). Shown are representative images and the mean MFI (± SD) of pY from three independent experiments. Bar, 2.5 µm. (G) Effect of SLO treatment on BCR internalization. B cells were incubated at 4°C with biotinylated F(ab′) 2 anti–mouse IgM+IgG (10 µg/ml), followed by 37°C incubation with or without SLO for the indicated times. Cells were then labeled with PE-streptavidin at 4°C and analyzed by flow cytometry to determine the percentage of surface-labeled BCRs remaining on the cell surface. Shown is the mean percentage (± SD) from three independent experiments. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P
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    Image Search Results


    JEV infection downregulates miR-432 levels and leads to SOCS5 upregulation. Human brain microglial cells were infected by JEV (MOI 5) and cells were harvested at various time points to determine miR-432 levels. (A) Graph showing downregulation of miR-432 levels at 24 and 48 hours post infection as compared to 12 hours sample. TaqMan probe specific to miR-432 were used to determine fold change. The C t values were normalized by RNU-24 levels. (B) Graph showing reduced levels of miR-432 in JEV infected mice brain. The RNA was isolated from brain tissue of 2 day and 4 day infected mice and mock infected mice brain was used as control. The C t values were normalized by RNU-6 levels. (C) Western blots depicting upregulation of SOCS5 upon JEV infection. CHME3 cells were infected by JEV (MOI 5) and harvested after 24 and 48 hours. The average fold change values with respect to control have been mentioned. (D) Western blots showing upregulation of SOCS5 in JEV infected brain tissue. Mock infected mice brain tissue was used as control. Infected mice were harvested 2 day and 4 day post infection. β-tubulin was used for normalization. The average fold change values with respect to control have been mentioned. (E) Immunohistochemistry image showing increased levels of SOCS5 protein in brain of JEV infected BALB/c mice. Mock and JEV infected mice brain sections were collected 2 days post JEV infection. Enhanced levels of Alexa 488 florescence was observed in JEV infected brain section (Magnification 20x, Scale bar 100 μm). All experiments were performed in triplicates. The data are shown as mean ± S.E from three independent experiments. The fold change is significant where *denotes P

    Journal: Scientific Reports

    Article Title: Japanese Encephalitis Virus exploits the microRNA-432 to regulate the expression of Suppressor of Cytokine Signaling (SOCS) 5

    doi: 10.1038/srep27685

    Figure Lengend Snippet: JEV infection downregulates miR-432 levels and leads to SOCS5 upregulation. Human brain microglial cells were infected by JEV (MOI 5) and cells were harvested at various time points to determine miR-432 levels. (A) Graph showing downregulation of miR-432 levels at 24 and 48 hours post infection as compared to 12 hours sample. TaqMan probe specific to miR-432 were used to determine fold change. The C t values were normalized by RNU-24 levels. (B) Graph showing reduced levels of miR-432 in JEV infected mice brain. The RNA was isolated from brain tissue of 2 day and 4 day infected mice and mock infected mice brain was used as control. The C t values were normalized by RNU-6 levels. (C) Western blots depicting upregulation of SOCS5 upon JEV infection. CHME3 cells were infected by JEV (MOI 5) and harvested after 24 and 48 hours. The average fold change values with respect to control have been mentioned. (D) Western blots showing upregulation of SOCS5 in JEV infected brain tissue. Mock infected mice brain tissue was used as control. Infected mice were harvested 2 day and 4 day post infection. β-tubulin was used for normalization. The average fold change values with respect to control have been mentioned. (E) Immunohistochemistry image showing increased levels of SOCS5 protein in brain of JEV infected BALB/c mice. Mock and JEV infected mice brain sections were collected 2 days post JEV infection. Enhanced levels of Alexa 488 florescence was observed in JEV infected brain section (Magnification 20x, Scale bar 100 μm). All experiments were performed in triplicates. The data are shown as mean ± S.E from three independent experiments. The fold change is significant where *denotes P

    Article Snippet: The sections were washed and incubated with Alexa 488 conjugated secondary antibody (A 11034, Life technologies) for 1 hour and washed.

    Techniques: Infection, Mouse Assay, Isolation, Western Blot, Immunohistochemistry

    ROS is produced in both prion-infected and GDL-exposed COCS. ( A ) COCS were exposed to POM1 for 8 h-10 days, and neuronal loss was assessed by NeuN + pixel coverage. Neuronal loss was significant at 3 dpe. Untreated slices (ut), or slices exposed to pooled IgG, were used as controls (grey). ( B ) In prion-infected COCS (+) neuronal loss became significant at 45 dpi. Controls (-): exposure to non-infectious brain homogenate. Data were analyzed using a two-tailed t-test; n = 9 biological replicates. ( C—D ) COCS were exposed to DHE, and the number of positive fluorescent particles was counted per 10x view field. ( C ) DHE conversion assays were performed at various time points between 1 h and 240 h. Data from three experiments are represented in this graph; n = 27 biological replicates. Slices that were untreated or exposed to pooled IgG were used as controls (grey). ROS production was detectable 4 hours after POM1 exposure and after 1 h with a higher POM1 concentration (335 nM). Here and henceforth data are presented as average ± s.d. and were analyzed by one-way ANOVA with Dunnett’s post-hoc test unless otherwise specified (***: p

    Journal: PLoS Pathogens

    Article Title: Prion Infections and Anti-PrP Antibodies Trigger Converging Neurotoxic Pathways

    doi: 10.1371/journal.ppat.1004662

    Figure Lengend Snippet: ROS is produced in both prion-infected and GDL-exposed COCS. ( A ) COCS were exposed to POM1 for 8 h-10 days, and neuronal loss was assessed by NeuN + pixel coverage. Neuronal loss was significant at 3 dpe. Untreated slices (ut), or slices exposed to pooled IgG, were used as controls (grey). ( B ) In prion-infected COCS (+) neuronal loss became significant at 45 dpi. Controls (-): exposure to non-infectious brain homogenate. Data were analyzed using a two-tailed t-test; n = 9 biological replicates. ( C—D ) COCS were exposed to DHE, and the number of positive fluorescent particles was counted per 10x view field. ( C ) DHE conversion assays were performed at various time points between 1 h and 240 h. Data from three experiments are represented in this graph; n = 27 biological replicates. Slices that were untreated or exposed to pooled IgG were used as controls (grey). ROS production was detectable 4 hours after POM1 exposure and after 1 h with a higher POM1 concentration (335 nM). Here and henceforth data are presented as average ± s.d. and were analyzed by one-way ANOVA with Dunnett’s post-hoc test unless otherwise specified (***: p

    Article Snippet: Secondary antibodies were horseradish peroxidase (HRP)-conjugated rabbit anti–mouse IgG1 (1:10,000, Zymed), and goat anti–rabbit IgG1 (1:10,000, Zymed).

    Techniques: Produced, Infection, Two Tailed Test, Concentration Assay

    ROS scavengers and calpain inhibitors are beneficial to prion-infected COCS and mice. ( A ) Lower graph: COCS were inoculated with RML or NBH, cultured for 21 dpi, and then treated with various compounds for 22 days. Upper graph: COCS were cultured for 14 days, incubated with POM1 and treated with the same compounds for 10–14 days. Viability (NeuN) was normalized to COCS exposed to IgG and to non-infectious brain homogenate (upper and lower gray dots, respectively). Calpain inhibitors (Calpeptin), antioxidants (Ascorbate, Isoascorbate, NaC), and superoxide dismutase mimetics (MnTBAP) were neuroprotective in both paradigms (red dots), whereas inhibition of AMPA, kainate receptors (CNQX), NMDA receptors (MK801), a mitochondrial membrane permeability transition pore (methazolamide), caspases (ZVAD), and of ER calcium stores (Dantrolene), (black dots) was ineffective; n = 9 biological replicates. The effects of compounds labeled with “†” on POM1-exposed COCS and zVAD labeled with “§” on RML-infected COCS were reported previously [ 15 ], [ 13 ] and are reproduced here for convenience. ( B ) COCS were exposed to NBH (-) or to RML (+) as in (A), and harvested at 39 dpi ( n = 3). Prion titers were determined by SCEPA. No reduction in prion infectivity was observed in COCS treated with ascorbate (Asc) or NaC ( n = 3 biological replicates). ( C ) Prion-infected COCS were treated with Asc. Protection was discernible for ≥53 dpi (red bars). Data were analyzed using a two-tailed t-test; n = 9 biological replicates. ( D ) Survival curves of prion-infected tg a 20 mice treated with AcHyT (median 95dpi) or vehicle (drinking water; median 88.5dpi). AcHyT treatment increased the incubation time of prion-infected mice (p = 0.0287). Results were analyzed using a Mantel-Cox log-rank test; n = 6 untreated mice and n = 7 AcHyT-treated mice. ( E ) COCS were treated with antioxidants from 21 dpi onwards ( n = 3) and harvested at 40 dpi. Cleaved α-fodrin bands (145 and 150 kDa) were quantified and normalized to GAPDH (right). None of the antioxidants suppressed the prion-induced enhancement of α-fodrin cleavage; n = 3 biological replicates. ( F ) COCS were cultured for 14 days, treated with scFvPOM1 (400nM) or scFvPOM1 preincubated with recombinant PrP (1200nM) in presence of ascorbate, and harvested at 3 dpe. Cleaved α-fodrin bands (145 and 150 kDa) were quantified and normalized to calnexin (right). Ascorbate did not suppress the GDL induced enhancement of α-fodrin cleavage; n = 3 biological replicates.

    Journal: PLoS Pathogens

    Article Title: Prion Infections and Anti-PrP Antibodies Trigger Converging Neurotoxic Pathways

    doi: 10.1371/journal.ppat.1004662

    Figure Lengend Snippet: ROS scavengers and calpain inhibitors are beneficial to prion-infected COCS and mice. ( A ) Lower graph: COCS were inoculated with RML or NBH, cultured for 21 dpi, and then treated with various compounds for 22 days. Upper graph: COCS were cultured for 14 days, incubated with POM1 and treated with the same compounds for 10–14 days. Viability (NeuN) was normalized to COCS exposed to IgG and to non-infectious brain homogenate (upper and lower gray dots, respectively). Calpain inhibitors (Calpeptin), antioxidants (Ascorbate, Isoascorbate, NaC), and superoxide dismutase mimetics (MnTBAP) were neuroprotective in both paradigms (red dots), whereas inhibition of AMPA, kainate receptors (CNQX), NMDA receptors (MK801), a mitochondrial membrane permeability transition pore (methazolamide), caspases (ZVAD), and of ER calcium stores (Dantrolene), (black dots) was ineffective; n = 9 biological replicates. The effects of compounds labeled with “†” on POM1-exposed COCS and zVAD labeled with “§” on RML-infected COCS were reported previously [ 15 ], [ 13 ] and are reproduced here for convenience. ( B ) COCS were exposed to NBH (-) or to RML (+) as in (A), and harvested at 39 dpi ( n = 3). Prion titers were determined by SCEPA. No reduction in prion infectivity was observed in COCS treated with ascorbate (Asc) or NaC ( n = 3 biological replicates). ( C ) Prion-infected COCS were treated with Asc. Protection was discernible for ≥53 dpi (red bars). Data were analyzed using a two-tailed t-test; n = 9 biological replicates. ( D ) Survival curves of prion-infected tg a 20 mice treated with AcHyT (median 95dpi) or vehicle (drinking water; median 88.5dpi). AcHyT treatment increased the incubation time of prion-infected mice (p = 0.0287). Results were analyzed using a Mantel-Cox log-rank test; n = 6 untreated mice and n = 7 AcHyT-treated mice. ( E ) COCS were treated with antioxidants from 21 dpi onwards ( n = 3) and harvested at 40 dpi. Cleaved α-fodrin bands (145 and 150 kDa) were quantified and normalized to GAPDH (right). None of the antioxidants suppressed the prion-induced enhancement of α-fodrin cleavage; n = 3 biological replicates. ( F ) COCS were cultured for 14 days, treated with scFvPOM1 (400nM) or scFvPOM1 preincubated with recombinant PrP (1200nM) in presence of ascorbate, and harvested at 3 dpe. Cleaved α-fodrin bands (145 and 150 kDa) were quantified and normalized to calnexin (right). Ascorbate did not suppress the GDL induced enhancement of α-fodrin cleavage; n = 3 biological replicates.

    Article Snippet: Secondary antibodies were horseradish peroxidase (HRP)-conjugated rabbit anti–mouse IgG1 (1:10,000, Zymed), and goat anti–rabbit IgG1 (1:10,000, Zymed).

    Techniques: Infection, Mouse Assay, Cell Culture, Incubation, Inhibition, Permeability, Labeling, Two Tailed Test, Recombinant

    anti-FT antibody POM2 counteracts prion-induced neurotoxicity in COCS. ( A ) COCS prepared from tg a 20 mice were exposed to NBH (gray) or infected with RML prions (white), treated with pooled IgG or POM2 (335nM), and stained for NeuN at 44dpi. POM2 treatment afforded significant neuroprotection against prion infection; n = 9 biological replicates. ( B ) Prion titers of slice homogenates treated as in (A) were determined by SCEPA. POM2 treatment did not significantly reduce infectivity titers in prion-infected COCS. Data points represent the log of the median infectivity dose per mg of tissue culture (TCID 50 mg -1 ) ± s.d.; n = 4 biological replicates. ( C ) COCS homogenates were treated as in (A), digested with proteinase K, and probed with the antibody POM1 for PrP Sc . While the PrP Sc bands between 18–30 kDa were somewhat decreased by POM2 treatment, there was a concomitant increase in higher-molecular PrP-immunoreactive moieties presumably representing SDS-resistant oligomers of PrP Sc . Because of the different electrophoretic patterns of POM2-treated samples, we have opted to perform a quantification of the entire lane (right panel). POM2 treatment did not decrease the load of PK-resistant PrP Sc ; n = 3 biological replicates.

    Journal: PLoS Pathogens

    Article Title: Prion Infections and Anti-PrP Antibodies Trigger Converging Neurotoxic Pathways

    doi: 10.1371/journal.ppat.1004662

    Figure Lengend Snippet: anti-FT antibody POM2 counteracts prion-induced neurotoxicity in COCS. ( A ) COCS prepared from tg a 20 mice were exposed to NBH (gray) or infected with RML prions (white), treated with pooled IgG or POM2 (335nM), and stained for NeuN at 44dpi. POM2 treatment afforded significant neuroprotection against prion infection; n = 9 biological replicates. ( B ) Prion titers of slice homogenates treated as in (A) were determined by SCEPA. POM2 treatment did not significantly reduce infectivity titers in prion-infected COCS. Data points represent the log of the median infectivity dose per mg of tissue culture (TCID 50 mg -1 ) ± s.d.; n = 4 biological replicates. ( C ) COCS homogenates were treated as in (A), digested with proteinase K, and probed with the antibody POM1 for PrP Sc . While the PrP Sc bands between 18–30 kDa were somewhat decreased by POM2 treatment, there was a concomitant increase in higher-molecular PrP-immunoreactive moieties presumably representing SDS-resistant oligomers of PrP Sc . Because of the different electrophoretic patterns of POM2-treated samples, we have opted to perform a quantification of the entire lane (right panel). POM2 treatment did not decrease the load of PK-resistant PrP Sc ; n = 3 biological replicates.

    Article Snippet: Secondary antibodies were horseradish peroxidase (HRP)-conjugated rabbit anti–mouse IgG1 (1:10,000, Zymed), and goat anti–rabbit IgG1 (1:10,000, Zymed).

    Techniques: Mouse Assay, Infection, Staining

    The transcriptome of RML-infected and POM1-treated COCS. ( A ) The number of genes upregulated or downregulated at various time points after RML infection (left) or exposure to POM1 (right). Upregulation or downregulation was defined as > 2-fold change over NBH-exposed (left) or pooled-IgG exposed (right) COCS at the same time point. In both paradigms, the total number of differentially expressed genes increased over time. ( B ) Overlap of upregulated (left) and downregulated (right) genes in RML-infected vs. POM1-exposed COCS. The two paradigms shared 80% of downregulated and 38% of upregulated genes when comparing 3dpe GDL and 45dpi RML. ( C ) Correlation coefficients (y-axis) of all genes and genes involved in specific signaling pathways between POM1 exposure at different time points (x-axis) and RML exposure at 45 dpi.

    Journal: PLoS Pathogens

    Article Title: Prion Infections and Anti-PrP Antibodies Trigger Converging Neurotoxic Pathways

    doi: 10.1371/journal.ppat.1004662

    Figure Lengend Snippet: The transcriptome of RML-infected and POM1-treated COCS. ( A ) The number of genes upregulated or downregulated at various time points after RML infection (left) or exposure to POM1 (right). Upregulation or downregulation was defined as > 2-fold change over NBH-exposed (left) or pooled-IgG exposed (right) COCS at the same time point. In both paradigms, the total number of differentially expressed genes increased over time. ( B ) Overlap of upregulated (left) and downregulated (right) genes in RML-infected vs. POM1-exposed COCS. The two paradigms shared 80% of downregulated and 38% of upregulated genes when comparing 3dpe GDL and 45dpi RML. ( C ) Correlation coefficients (y-axis) of all genes and genes involved in specific signaling pathways between POM1 exposure at different time points (x-axis) and RML exposure at 45 dpi.

    Article Snippet: Secondary antibodies were horseradish peroxidase (HRP)-conjugated rabbit anti–mouse IgG1 (1:10,000, Zymed), and goat anti–rabbit IgG1 (1:10,000, Zymed).

    Techniques: Infection

    ER stress is detected in both prion-infected and GDL-exposed COCS. ( A ) Tg a 20 COCS (prion-infected or NBH-exposed) were harvested at 42 dpi, and probed for p-PERK, p-eIF2α, and ATF4 by western blot. Densitometry (normalized to actin) showed a trend towards increased p-PERK ( p = 0.14) and significantly elevated p-eIF2α and ATF4 after prion infection. ( B ) COCS were cultured for 14 days, treated with POM1 or IgG, harvested at 3 dpe, and probed for p-PERK, p-eIF2α and ATF4 by western blot. Densitometry (normalized to actin) revealed increased p-PERK, p-eIF2α, and ATF4. Densitometry data were analyzed using a two-tailed t-test; n = 3 biological replicates.

    Journal: PLoS Pathogens

    Article Title: Prion Infections and Anti-PrP Antibodies Trigger Converging Neurotoxic Pathways

    doi: 10.1371/journal.ppat.1004662

    Figure Lengend Snippet: ER stress is detected in both prion-infected and GDL-exposed COCS. ( A ) Tg a 20 COCS (prion-infected or NBH-exposed) were harvested at 42 dpi, and probed for p-PERK, p-eIF2α, and ATF4 by western blot. Densitometry (normalized to actin) showed a trend towards increased p-PERK ( p = 0.14) and significantly elevated p-eIF2α and ATF4 after prion infection. ( B ) COCS were cultured for 14 days, treated with POM1 or IgG, harvested at 3 dpe, and probed for p-PERK, p-eIF2α and ATF4 by western blot. Densitometry (normalized to actin) revealed increased p-PERK, p-eIF2α, and ATF4. Densitometry data were analyzed using a two-tailed t-test; n = 3 biological replicates.

    Article Snippet: Secondary antibodies were horseradish peroxidase (HRP)-conjugated rabbit anti–mouse IgG1 (1:10,000, Zymed), and goat anti–rabbit IgG1 (1:10,000, Zymed).

    Techniques: Infection, Western Blot, Cell Culture, Two Tailed Test

    The number fraction of rAb-MP aggregates to all particles as a function of goat IgG concentration in PBS containing 0.1% BSA. Particle counts were obtained from bright field microscope images. The standard deviation was calculated from three replicates.

    Journal: PLoS ONE

    Article Title: A Versatile Microparticle-Based Immunoaggregation Assay for Macromolecular Biomarker Detection and Quantification

    doi: 10.1371/journal.pone.0115046

    Figure Lengend Snippet: The number fraction of rAb-MP aggregates to all particles as a function of goat IgG concentration in PBS containing 0.1% BSA. Particle counts were obtained from bright field microscope images. The standard deviation was calculated from three replicates.

    Article Snippet: Materials and Methods Streptavidin–functionalized Microparticle (MP) (Dynabeads M-280 with a diameter of 2.8 μm), biotinylated polyclonal rabbit anti-goat IgG (rAb) and goat anti-rabbit IgG (goat IgG) (labeled with Alexa Fluor 488) were bought from Life Technologies (Carlsbad, CA, USA).

    Techniques: Concentration Assay, Microscopy, Standard Deviation

    Accusizer measurement results for (A) FITC labeled rAb-MP without goat Ig G and (B) FITC labeled rAb-MP with 36 ng/mL goat IgG as a model macromolecular biomarker. The concentration of rAb-MP was kept constant at 53.4 μg/mL.

    Journal: PLoS ONE

    Article Title: A Versatile Microparticle-Based Immunoaggregation Assay for Macromolecular Biomarker Detection and Quantification

    doi: 10.1371/journal.pone.0115046

    Figure Lengend Snippet: Accusizer measurement results for (A) FITC labeled rAb-MP without goat Ig G and (B) FITC labeled rAb-MP with 36 ng/mL goat IgG as a model macromolecular biomarker. The concentration of rAb-MP was kept constant at 53.4 μg/mL.

    Article Snippet: Materials and Methods Streptavidin–functionalized Microparticle (MP) (Dynabeads M-280 with a diameter of 2.8 μm), biotinylated polyclonal rabbit anti-goat IgG (rAb) and goat anti-rabbit IgG (goat IgG) (labeled with Alexa Fluor 488) were bought from Life Technologies (Carlsbad, CA, USA).

    Techniques: Labeling, Biomarker Assay, Concentration Assay

    Fluorescence Microscope images: (A) FITC labeled rAb-MP without goat Ig G and (B) FITC labeled rAb-MP with 36 ng/mL goat IgG as a model biomarker. The concentration of rAb-MP was kept constant at 53.4 μg/mL.

    Journal: PLoS ONE

    Article Title: A Versatile Microparticle-Based Immunoaggregation Assay for Macromolecular Biomarker Detection and Quantification

    doi: 10.1371/journal.pone.0115046

    Figure Lengend Snippet: Fluorescence Microscope images: (A) FITC labeled rAb-MP without goat Ig G and (B) FITC labeled rAb-MP with 36 ng/mL goat IgG as a model biomarker. The concentration of rAb-MP was kept constant at 53.4 μg/mL.

    Article Snippet: Materials and Methods Streptavidin–functionalized Microparticle (MP) (Dynabeads M-280 with a diameter of 2.8 μm), biotinylated polyclonal rabbit anti-goat IgG (rAb) and goat anti-rabbit IgG (goat IgG) (labeled with Alexa Fluor 488) were bought from Life Technologies (Carlsbad, CA, USA).

    Techniques: Fluorescence, Microscopy, Labeling, Biomarker Assay, Concentration Assay

    Western blot of E. histolytica rPPDK and native PPDK using anti-rPPDK IgG and anti-EhESA IgG polyclonal antibodies.

    Journal: Pathogens and Global Health

    Article Title: Development and initial evaluation of a lateral flow dipstick test for antigen detection of Entamoeba histolytica in stool sample

    doi: 10.1080/20477724.2017.1300421

    Figure Lengend Snippet: Western blot of E. histolytica rPPDK and native PPDK using anti-rPPDK IgG and anti-EhESA IgG polyclonal antibodies.

    Article Snippet: The test line comprised anti-rPPDK IgG and the control line comprised polyclonal goat anti-rabbit IgG (Thermo Scientific) at 0.2 mg ml−1 .

    Techniques: Western Blot

    Binding of neuromyelitis optica (NMO)–immunoglobulin G (IgG) to human astrocyte cell lines (A, B) Fixed cells were incubated with NMO-IgG (pooled from patient sera), control IgG (pooled from healthy donor sera), and commercial aquaporin-4 (AQP4) IgG (rabbit polyclonal, cytoplasmic epitopes). AlexaFluor 488 goat anti-human IgG was used to probe for bound NMO-IgG and control IgG, and AlexaFluor 594 goat anti-rabbit IgG to probe for commercial AQP4 IgG. (A: A4 cells; B: A cells). Nuclear counterstaining with TO-PRO-3 demonstrates the presence of cells in B. Scale bar, 10 μm. (C) Exposure of living A4 cells to NMO-IgG, but not to control IgG, leads to clustering/internalization of surface AQP4. Counterstaining with Phalloidin Alexa 488, after fixing and permeabilization, shows filamentous actin. Scale bar, 10 μm.

    Journal: Neurology® Neuroimmunology & Neuroinflammation

    Article Title: Effects of neuromyelitis optica–IgG at the blood–brain barrier in vitro

    doi: 10.1212/NXI.0000000000000311

    Figure Lengend Snippet: Binding of neuromyelitis optica (NMO)–immunoglobulin G (IgG) to human astrocyte cell lines (A, B) Fixed cells were incubated with NMO-IgG (pooled from patient sera), control IgG (pooled from healthy donor sera), and commercial aquaporin-4 (AQP4) IgG (rabbit polyclonal, cytoplasmic epitopes). AlexaFluor 488 goat anti-human IgG was used to probe for bound NMO-IgG and control IgG, and AlexaFluor 594 goat anti-rabbit IgG to probe for commercial AQP4 IgG. (A: A4 cells; B: A cells). Nuclear counterstaining with TO-PRO-3 demonstrates the presence of cells in B. Scale bar, 10 μm. (C) Exposure of living A4 cells to NMO-IgG, but not to control IgG, leads to clustering/internalization of surface AQP4. Counterstaining with Phalloidin Alexa 488, after fixing and permeabilization, shows filamentous actin. Scale bar, 10 μm.

    Article Snippet: As secondary antibodies, we used AlexaFluor 488 or 594 goat anti-rabbit IgG (Life Technologies: A-11008), AlexaFluor 488 goat anti-mouse IgG (Life Technologies: A-11001), and AlexaFluor 488 goat anti-human IgG (Life Technologies: A-11013).

    Techniques: Binding Assay, Incubation

    GIMAP6 expression and protein distribution in T lymphocytes. A , cell lines representing the various different hematopoietic lineages, including T lymphocytes (Jurkat), promyeloblasts (HL-60), monocytes (U-937), lymphoblasts (K-562), and B lymphocytes (Raji and Ramos), as well as cell lines derived from colon (HCT-15), kidney (293T), liver (Huh-7), and lung (A549 and H1299), were analyzed for GIMAP6 protein expression by immunoblot analysis. The amount of protein loaded onto each lane was estimated by anti-β-actin or anti-GAPDH. B , GIMAP6 RNA expression levels in the hematopoietic cell lines were analyzed by quantitative PCR assay. The results were normalized against the expression level of HPRT1. Error bars indicate standard deviation. C , GIMAP6 RNA expression in three subsets of human PBMCs: CD3+/CD4+, CD3+/CD8+, and CD3− cells. These were analyzed by quantitative PCR assay. The PBMCs were obtained from a healthy donor. HPRT1 was used to normalize the dataset. Error bars indicate standard deviation. D and E , cellular distribution of endogenous GIMAP6 in Jurkat T cells. Immunoblot analysis of various protein preparations, including that of the total cell lysate ( Input ), revealed that endogenous GIMAP6 is restricted to the cytosolic fraction of Jurkat T lymphocyte lysates ( D ). E , confocal laser-scanning microscopy showed that GIMAP6 ( purple , Alexa Fluor 647 goat anti-rabbit IgG) co-localized with the cytosolic marker GAPDH ( green , Alexa Fluor 488 goat anti-mouse IgG). DAPI ( blue ) was used as a nuclear tracking dye. F , cellular distribution of endogenous GIMAP6 in primary CD3+ T cells. Primary CD3+ T cells enriched from PBMCs were used to perform immunofluorescence microscopy, which showed that GIMAP6 ( green , Alexa Fluor 488 goat anti-rabbit IgG) co-localized with the cytosolic marker GAPDH ( red , Alexa Fluor 594 goat anti-mouse IgG). DAPI ( blue ) was used as a nuclear tracking dye.

    Journal: The Journal of Biological Chemistry

    Article Title: Functional and biochemical characterization of a T cell-associated anti-apoptotic protein, GIMAP6

    doi: 10.1074/jbc.M116.768689

    Figure Lengend Snippet: GIMAP6 expression and protein distribution in T lymphocytes. A , cell lines representing the various different hematopoietic lineages, including T lymphocytes (Jurkat), promyeloblasts (HL-60), monocytes (U-937), lymphoblasts (K-562), and B lymphocytes (Raji and Ramos), as well as cell lines derived from colon (HCT-15), kidney (293T), liver (Huh-7), and lung (A549 and H1299), were analyzed for GIMAP6 protein expression by immunoblot analysis. The amount of protein loaded onto each lane was estimated by anti-β-actin or anti-GAPDH. B , GIMAP6 RNA expression levels in the hematopoietic cell lines were analyzed by quantitative PCR assay. The results were normalized against the expression level of HPRT1. Error bars indicate standard deviation. C , GIMAP6 RNA expression in three subsets of human PBMCs: CD3+/CD4+, CD3+/CD8+, and CD3− cells. These were analyzed by quantitative PCR assay. The PBMCs were obtained from a healthy donor. HPRT1 was used to normalize the dataset. Error bars indicate standard deviation. D and E , cellular distribution of endogenous GIMAP6 in Jurkat T cells. Immunoblot analysis of various protein preparations, including that of the total cell lysate ( Input ), revealed that endogenous GIMAP6 is restricted to the cytosolic fraction of Jurkat T lymphocyte lysates ( D ). E , confocal laser-scanning microscopy showed that GIMAP6 ( purple , Alexa Fluor 647 goat anti-rabbit IgG) co-localized with the cytosolic marker GAPDH ( green , Alexa Fluor 488 goat anti-mouse IgG). DAPI ( blue ) was used as a nuclear tracking dye. F , cellular distribution of endogenous GIMAP6 in primary CD3+ T cells. Primary CD3+ T cells enriched from PBMCs were used to perform immunofluorescence microscopy, which showed that GIMAP6 ( green , Alexa Fluor 488 goat anti-rabbit IgG) co-localized with the cytosolic marker GAPDH ( red , Alexa Fluor 594 goat anti-mouse IgG). DAPI ( blue ) was used as a nuclear tracking dye.

    Article Snippet: Alexa Fluor 647 or 488 goat anti-rabbit IgG (Life Technologies) and Alexa Fluor 488 or 594 goat anti-mouse IgG (Life Technologies) was used.

    Techniques: Expressing, Derivative Assay, RNA Expression, Real-time Polymerase Chain Reaction, Standard Deviation, Confocal Laser Scanning Microscopy, Marker, Immunofluorescence, Microscopy

    SLO injury of the PM reduces BCR activation and internalization. (A–D) BCR–CTB coclustering in the presence or absence of SLO. B cells were incubated at 4°C with Cy3-Fab (5 µg/ml; −XL) or AF546-F(ab′) 2 anti–mouse IgM+IgG (5 µg/ml; +XL) plus AF488-CTB (3 µg/ml) and warmed to 37°C for 5 min with or without SLO, followed by fixation and confocal microscopy. (A) Representative images. Bar, 2.5 µm. (B) Percentages (± SD) of all B cells exhibiting polarized BCR clusters (capping). (C) Percentages of B cells with polarized BCR clusters showing impaired BCR caps. (D) Percentages of B cells with polarized BCR clusters showing BCR–CTB coclustering. Data in B–D were generated by visual inspection of images from four independent experiments. (E and F) Tyrosine phosphorylation (pY) of B cells treated with or without SLO. B cells were incubated at 4°C with SLO and AF546-F(ab′) 2 -goat anti–mouse IgM+IgG (5 µg/ml) and warmed to 37°C for 5 min, followed by fixation, permeabilization, and staining for phosphotyrosine (pY) and analysis of confocal microscopy (E) and flow cytometry (F). Shown are representative images and the mean MFI (± SD) of pY from three independent experiments. Bar, 2.5 µm. (G) Effect of SLO treatment on BCR internalization. B cells were incubated at 4°C with biotinylated F(ab′) 2 anti–mouse IgM+IgG (10 µg/ml), followed by 37°C incubation with or without SLO for the indicated times. Cells were then labeled with PE-streptavidin at 4°C and analyzed by flow cytometry to determine the percentage of surface-labeled BCRs remaining on the cell surface. Shown is the mean percentage (± SD) from three independent experiments. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P

    Journal: The Journal of Cell Biology

    Article Title: Lipid raft–dependent plasma membrane repair interferes with the activation of B lymphocytes

    doi: 10.1083/jcb.201505030

    Figure Lengend Snippet: SLO injury of the PM reduces BCR activation and internalization. (A–D) BCR–CTB coclustering in the presence or absence of SLO. B cells were incubated at 4°C with Cy3-Fab (5 µg/ml; −XL) or AF546-F(ab′) 2 anti–mouse IgM+IgG (5 µg/ml; +XL) plus AF488-CTB (3 µg/ml) and warmed to 37°C for 5 min with or without SLO, followed by fixation and confocal microscopy. (A) Representative images. Bar, 2.5 µm. (B) Percentages (± SD) of all B cells exhibiting polarized BCR clusters (capping). (C) Percentages of B cells with polarized BCR clusters showing impaired BCR caps. (D) Percentages of B cells with polarized BCR clusters showing BCR–CTB coclustering. Data in B–D were generated by visual inspection of images from four independent experiments. (E and F) Tyrosine phosphorylation (pY) of B cells treated with or without SLO. B cells were incubated at 4°C with SLO and AF546-F(ab′) 2 -goat anti–mouse IgM+IgG (5 µg/ml) and warmed to 37°C for 5 min, followed by fixation, permeabilization, and staining for phosphotyrosine (pY) and analysis of confocal microscopy (E) and flow cytometry (F). Shown are representative images and the mean MFI (± SD) of pY from three independent experiments. Bar, 2.5 µm. (G) Effect of SLO treatment on BCR internalization. B cells were incubated at 4°C with biotinylated F(ab′) 2 anti–mouse IgM+IgG (10 µg/ml), followed by 37°C incubation with or without SLO for the indicated times. Cells were then labeled with PE-streptavidin at 4°C and analyzed by flow cytometry to determine the percentage of surface-labeled BCRs remaining on the cell surface. Shown is the mean percentage (± SD) from three independent experiments. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P

    Article Snippet: Analyses of lysosomal exocytosis B cells were incubated without or with SLO (200 ng/ml) for 5 min at 4°C and then 37°C for 5 min. To stain LIMP2 at the cell surface, cells were transferred to 4°C, incubated with rabbit anti-LIMP2 antibody (5 µg/ml; Sigma-Aldrich), followed by AF488 goat anti–rabbit IgG (5 µg/ml; Invitrogen) as secondary antibody and Cy3-Fab-goat anti–mouse IgM+IgG (5 µg/ml; Jackson ImmunoResearch Laboratories) for marking BCRs at 4°C without permeabilization.

    Techniques: Activation Assay, CtB Assay, Incubation, Confocal Microscopy, Generated, Staining, Flow Cytometry, Cytometry, Labeling

    PM wounding by SLO increases endocytosis. (A) Confocal fluorescence microscopy images of Texas red–dextran and cholera toxin B subunit (CTB) in B cells. Cells were stained with Alexa Fluor (AF) 488–CTB (3 µg/ml) at 4°C and incubated with or without SLO at 37°C in the presence of dextran (2.5 mg/ml) for 3 min. Cells were then washed and stained with AF405 goat anti–mouse IgG to label surface BCR at 4°C. Bar, 2.5 µm. (B) Percentage (± SD) of B cells (treated or not with SLO or SM) showing internalized dextran, determined by visual inspection of confocal images from three independent experiments. (C) Correlation coefficients of dextran and CTB staining in the presence or absence of SLO, determined by confocal fluorescence microscopy. Shown is the mean (± SD) of three independent experiments. (D and E) Quantification of CTB endocytosis by flow cytometry. B cells were labeled with AF488-CTB (1 µg/ml) at 4°C and treated with SLO at 37°C for 3 min. The surface CTB was quenched with anti-AF488 antibodies at 4°C before and after SLO treatment. The MFI of CTB was quantified by flow cytometry. Shown are a representative histogram (D) and the percentage (± SD) of internalized CTB from three independent experiments (E). (F and G) TEM analysis of CTB endocytosis. B cells were incubated at 4°C with biotin-CTB (2 µg/ml) followed by gold-streptavidin (10 nm, 2 µg/ml), and then treated with SLO for 1 and 5 min at 37°C. Shown are representative images (F) and the mean percentage (± SD) of internalized CTB gold particles from > 16 randomly selected cell profiles per condition from two independent experiments (G). Bar, 100 nm. **, P

    Journal: The Journal of Cell Biology

    Article Title: Lipid raft–dependent plasma membrane repair interferes with the activation of B lymphocytes

    doi: 10.1083/jcb.201505030

    Figure Lengend Snippet: PM wounding by SLO increases endocytosis. (A) Confocal fluorescence microscopy images of Texas red–dextran and cholera toxin B subunit (CTB) in B cells. Cells were stained with Alexa Fluor (AF) 488–CTB (3 µg/ml) at 4°C and incubated with or without SLO at 37°C in the presence of dextran (2.5 mg/ml) for 3 min. Cells were then washed and stained with AF405 goat anti–mouse IgG to label surface BCR at 4°C. Bar, 2.5 µm. (B) Percentage (± SD) of B cells (treated or not with SLO or SM) showing internalized dextran, determined by visual inspection of confocal images from three independent experiments. (C) Correlation coefficients of dextran and CTB staining in the presence or absence of SLO, determined by confocal fluorescence microscopy. Shown is the mean (± SD) of three independent experiments. (D and E) Quantification of CTB endocytosis by flow cytometry. B cells were labeled with AF488-CTB (1 µg/ml) at 4°C and treated with SLO at 37°C for 3 min. The surface CTB was quenched with anti-AF488 antibodies at 4°C before and after SLO treatment. The MFI of CTB was quantified by flow cytometry. Shown are a representative histogram (D) and the percentage (± SD) of internalized CTB from three independent experiments (E). (F and G) TEM analysis of CTB endocytosis. B cells were incubated at 4°C with biotin-CTB (2 µg/ml) followed by gold-streptavidin (10 nm, 2 µg/ml), and then treated with SLO for 1 and 5 min at 37°C. Shown are representative images (F) and the mean percentage (± SD) of internalized CTB gold particles from > 16 randomly selected cell profiles per condition from two independent experiments (G). Bar, 100 nm. **, P

    Article Snippet: Analyses of lysosomal exocytosis B cells were incubated without or with SLO (200 ng/ml) for 5 min at 4°C and then 37°C for 5 min. To stain LIMP2 at the cell surface, cells were transferred to 4°C, incubated with rabbit anti-LIMP2 antibody (5 µg/ml; Sigma-Aldrich), followed by AF488 goat anti–rabbit IgG (5 µg/ml; Invitrogen) as secondary antibody and Cy3-Fab-goat anti–mouse IgM+IgG (5 µg/ml; Jackson ImmunoResearch Laboratories) for marking BCRs at 4°C without permeabilization.

    Techniques: Fluorescence, Microscopy, CtB Assay, Staining, Incubation, Flow Cytometry, Cytometry, Labeling, Transmission Electron Microscopy