lectin ricinus communis agglutinin 1 rca 1  (Vector Laboratories)


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    Vector Laboratories lectin ricinus communis agglutinin 1 rca 1
    Lectin Ricinus Communis Agglutinin 1 Rca 1, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    biotinylated lectin ricinus communis agglutinin 1  (Vector Laboratories)


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    Vector Laboratories biotinylated lectin ricinus communis agglutinin 1
    Immunohistochemical illustration of Ricinus communis <t>agglutinin-1</t> <t>(RCA-1)</t> -positive microglial cells in the white matter of spinal cord . The RCA-1-positive microglial cells (arrowheads) in both healthy (A) and AvIDP-affected (B) chicken show incospicuous ramified microglial cells. Activated microglial cells or an increased number were not observed. A, B: asterisk = endothelium (also stains RCA-1 positive); scale bar = 250 μm.
    Biotinylated Lectin Ricinus Communis Agglutinin 1, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Acute paretic syndrome in juvenile White Leghorn chickens resembles late stages of acute inflammatory demyelinating polyneuropathies in humans"

    Article Title: Acute paretic syndrome in juvenile White Leghorn chickens resembles late stages of acute inflammatory demyelinating polyneuropathies in humans

    Journal: Journal of Neuroinflammation

    doi: 10.1186/1742-2094-7-7

    Immunohistochemical illustration of Ricinus communis agglutinin-1 (RCA-1) -positive microglial cells in the white matter of spinal cord . The RCA-1-positive microglial cells (arrowheads) in both healthy (A) and AvIDP-affected (B) chicken show incospicuous ramified microglial cells. Activated microglial cells or an increased number were not observed. A, B: asterisk = endothelium (also stains RCA-1 positive); scale bar = 250 μm.
    Figure Legend Snippet: Immunohistochemical illustration of Ricinus communis agglutinin-1 (RCA-1) -positive microglial cells in the white matter of spinal cord . The RCA-1-positive microglial cells (arrowheads) in both healthy (A) and AvIDP-affected (B) chicken show incospicuous ramified microglial cells. Activated microglial cells or an increased number were not observed. A, B: asterisk = endothelium (also stains RCA-1 positive); scale bar = 250 μm.

    Techniques Used: Immunohistochemical staining

    lectin ricinus communis agglutinin 1 rca 1  (Vector Laboratories)


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    Vector Laboratories lectin ricinus communis agglutinin 1 rca 1
    Lectin Ricinus Communis Agglutinin 1 Rca 1, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ricinus communis agglutinin i rca 1 lectins  (Vector Laboratories)


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    Vector Laboratories ricinus communis agglutinin i rca 1 lectins
    Ricinus Communis Agglutinin I Rca 1 Lectins, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ricinus communis agglutinin i rca 1 lectins  (Vector Laboratories)


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    Vector Laboratories ricinus communis agglutinin i rca 1 lectins
    Viperid snake venoms induces platelet desialylation. ( A , B ) Effect of the venoms from D. siamensis (DSV), A. halys (AHV) and B. multicinctus (BMV) on <t>RCA-1</t> binding. Effect of OSGE and oseltamivir on ( C ) DSV and ( D ) AHV venoms induced RCA-1 binding. Statistical analysis was performed with the one-way ANOVA test. n = 3. *** p < 0.001.
    Ricinus Communis Agglutinin I Rca 1 Lectins, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Platelet Desialylation Is a Novel Mechanism and Therapeutic Target in Daboia siamensis and Agkistrodon halys Envenomation-Induced Thrombocytopenia"

    Article Title: Platelet Desialylation Is a Novel Mechanism and Therapeutic Target in Daboia siamensis and Agkistrodon halys Envenomation-Induced Thrombocytopenia

    Journal: Molecules

    doi: 10.3390/molecules27227779

    Viperid snake venoms induces platelet desialylation. ( A , B ) Effect of the venoms from D. siamensis (DSV), A. halys (AHV) and B. multicinctus (BMV) on RCA-1 binding. Effect of OSGE and oseltamivir on ( C ) DSV and ( D ) AHV venoms induced RCA-1 binding. Statistical analysis was performed with the one-way ANOVA test. n = 3. *** p < 0.001.
    Figure Legend Snippet: Viperid snake venoms induces platelet desialylation. ( A , B ) Effect of the venoms from D. siamensis (DSV), A. halys (AHV) and B. multicinctus (BMV) on RCA-1 binding. Effect of OSGE and oseltamivir on ( C ) DSV and ( D ) AHV venoms induced RCA-1 binding. Statistical analysis was performed with the one-way ANOVA test. n = 3. *** p < 0.001.

    Techniques Used: Binding Assay

    lectin ricinus communis agglutinin 1  (Vector Laboratories)


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    Vector Laboratories lectin ricinus communis agglutinin 1
    Graphs represent the qualitative analysis (cells/mm 2 ) of activated microglia (( A <t>)—RCA-1</t> positive microglia; ( B )—Mac-3 positive microglia, ( C )—proliferating RCA-1/Ki-67 positive microglia), and GFAP positive astrocytes ( D ) during early remyelination (week 5.5) in the corpus callosum of animals treated with different dosages of CDP-choline or sham (PBS). The astrogliosis was not affected by CDP-choline treatment. However, the number of activated microglia in all animals treated with CDP-choline, regardless of the dose used, was significantly lower than in the control group. Significant effects between mice treated with different dosages of CDP-choline and PBS are indicated by asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001, n = 6).
    Lectin Ricinus Communis Agglutinin 1, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Regenerative Effects of CDP-Choline: A Dose-Dependent Study in the Toxic Cuprizone Model of De- and Remyelination"

    Article Title: Regenerative Effects of CDP-Choline: A Dose-Dependent Study in the Toxic Cuprizone Model of De- and Remyelination

    Journal: Pharmaceuticals

    doi: 10.3390/ph14111156

    Graphs represent the qualitative analysis (cells/mm 2 ) of activated microglia (( A )—RCA-1 positive microglia; ( B )—Mac-3 positive microglia, ( C )—proliferating RCA-1/Ki-67 positive microglia), and GFAP positive astrocytes ( D ) during early remyelination (week 5.5) in the corpus callosum of animals treated with different dosages of CDP-choline or sham (PBS). The astrogliosis was not affected by CDP-choline treatment. However, the number of activated microglia in all animals treated with CDP-choline, regardless of the dose used, was significantly lower than in the control group. Significant effects between mice treated with different dosages of CDP-choline and PBS are indicated by asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001, n = 6).
    Figure Legend Snippet: Graphs represent the qualitative analysis (cells/mm 2 ) of activated microglia (( A )—RCA-1 positive microglia; ( B )—Mac-3 positive microglia, ( C )—proliferating RCA-1/Ki-67 positive microglia), and GFAP positive astrocytes ( D ) during early remyelination (week 5.5) in the corpus callosum of animals treated with different dosages of CDP-choline or sham (PBS). The astrogliosis was not affected by CDP-choline treatment. However, the number of activated microglia in all animals treated with CDP-choline, regardless of the dose used, was significantly lower than in the control group. Significant effects between mice treated with different dosages of CDP-choline and PBS are indicated by asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001, n = 6).

    Techniques Used:

    fluorescein ricinus communis agglutinin 1 lectin  (Vector Laboratories)


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    Vector Laboratories fluorescein ricinus communis agglutinin 1 lectin

    Fluorescein Ricinus Communis Agglutinin 1 Lectin, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Protocol for induction and characterization of microglia extracellular traps in murine and human microglia cells"

    Article Title: Protocol for induction and characterization of microglia extracellular traps in murine and human microglia cells

    Journal: STAR Protocols

    doi: 10.1016/j.xpro.2021.100678


    Figure Legend Snippet:

    Techniques Used: Recombinant, Staining, Plasmid Preparation, Software, Phagocytosis Assay, Microscopy

    rhodamine labeled ricinus communis agglutinin i (rca i, rca120)  (Vector Laboratories)


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    Vector Laboratories rhodamine labeled ricinus communis agglutinin i (rca i, rca120)
    Rhodamine Labeled Ricinus Communis Agglutinin I (Rca I, Rca120), supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ricinus communis agglutinin 1 rca 1 lectin  (Vector Laboratories)


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    Vector Laboratories ricinus communis agglutinin 1 rca 1 lectin
    NLRP12 inhibition regulates cellular proliferation. ( a ) NLRP12 expression was observed in glioma (LN18) and microglial (BV2) cells using immunofluorescence (magnification: 40X, scale bar: 50 μm). ( b ) Colony formation assay shows increased cellular proliferation in the NLRP12si treated microglia. Magnification: 40X. ( c , d ) Quantification of both BV2 and LN18 cells per colony. ( e ) Effect of LN18 (NLRP12 siRNA treated) cell conditioned medium (CM) on BV2 cell migration was assessed using migration assay. 8 image sections per sample were quantified for migration analysis. Student’s t-test and one-way AVOVA were performed to find the statistical significance of colony formation and migration assay respectively (p-value; *<0.05, **<0.005). The error bar indicates standard error of mean. ( f ) NLRP12 expression in human normal brain tissue and glioma sections, using anti-NLRP12 antibody (N = 7). Nuclei were stained blue with DAPI and microglia were stained green with FITC tagged lectin RCA (Ricinus Communis <t>Agglutinin-1);</t> Magnification: 20X, scale bar: 100 µm.
    Ricinus Communis Agglutinin 1 Rca 1 Lectin, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Differential Expression Profile of NLRs and AIM2 in Glioma and Implications for NLRP12 in Glioblastoma"

    Article Title: Differential Expression Profile of NLRs and AIM2 in Glioma and Implications for NLRP12 in Glioblastoma

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-44854-4

    NLRP12 inhibition regulates cellular proliferation. ( a ) NLRP12 expression was observed in glioma (LN18) and microglial (BV2) cells using immunofluorescence (magnification: 40X, scale bar: 50 μm). ( b ) Colony formation assay shows increased cellular proliferation in the NLRP12si treated microglia. Magnification: 40X. ( c , d ) Quantification of both BV2 and LN18 cells per colony. ( e ) Effect of LN18 (NLRP12 siRNA treated) cell conditioned medium (CM) on BV2 cell migration was assessed using migration assay. 8 image sections per sample were quantified for migration analysis. Student’s t-test and one-way AVOVA were performed to find the statistical significance of colony formation and migration assay respectively (p-value; *<0.05, **<0.005). The error bar indicates standard error of mean. ( f ) NLRP12 expression in human normal brain tissue and glioma sections, using anti-NLRP12 antibody (N = 7). Nuclei were stained blue with DAPI and microglia were stained green with FITC tagged lectin RCA (Ricinus Communis Agglutinin-1); Magnification: 20X, scale bar: 100 µm.
    Figure Legend Snippet: NLRP12 inhibition regulates cellular proliferation. ( a ) NLRP12 expression was observed in glioma (LN18) and microglial (BV2) cells using immunofluorescence (magnification: 40X, scale bar: 50 μm). ( b ) Colony formation assay shows increased cellular proliferation in the NLRP12si treated microglia. Magnification: 40X. ( c , d ) Quantification of both BV2 and LN18 cells per colony. ( e ) Effect of LN18 (NLRP12 siRNA treated) cell conditioned medium (CM) on BV2 cell migration was assessed using migration assay. 8 image sections per sample were quantified for migration analysis. Student’s t-test and one-way AVOVA were performed to find the statistical significance of colony formation and migration assay respectively (p-value; *<0.05, **<0.005). The error bar indicates standard error of mean. ( f ) NLRP12 expression in human normal brain tissue and glioma sections, using anti-NLRP12 antibody (N = 7). Nuclei were stained blue with DAPI and microglia were stained green with FITC tagged lectin RCA (Ricinus Communis Agglutinin-1); Magnification: 20X, scale bar: 100 µm.

    Techniques Used: Inhibition, Expressing, Immunofluorescence, Colony Assay, Migration, Staining

    biotinylated lectin ricinus communis agglutinin i rca 1  (Vector Laboratories)


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    Vector Laboratories biotinylated lectin ricinus communis agglutinin i rca 1
    β1,4GalTV regulates gliomagenesis and the transdifferentiation of glioma stem-like cells into endothelial cells depending on its galactosylation activity. A, total lysates of T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G) were analyzed with an <t>RCA-1</t> lectin blot. The protein expression of GAPDH served as a loading control. B, nude mice were injected with T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). Four weeks later, photos were taken for the xenograft. Scale bars = 2 mm. C, Kaplan–Meier survival curves of nude mice injected with T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). D, immunofluorescence analysis shows co-localization of the human endothelial cell marker CD31 and the tumor cell marker GFP in tumor xenografts formed by T698968 cells expressing GFP and LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). Scale bars = 10 μm. E, the relative vascular density derived from glioma stem-like cells was quantified. Results are expressed as mean ± S.E. (n = 3; *, p < 0.05; **, p < 0.01). F, representative images of tube formation derived from T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). Scale bars = 20 μm. G, quantification of mean tube length in F. The results are expressed as mean ± S.E. (n = 3; ***, p < 0.001).
    Biotinylated Lectin Ricinus Communis Agglutinin I Rca 1, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "β1,4-Galactosyltransferase V activates Notch1 signaling in glioma stem-like cells and promotes their transdifferentiation into endothelial cells"

    Article Title: β1,4-Galactosyltransferase V activates Notch1 signaling in glioma stem-like cells and promotes their transdifferentiation into endothelial cells

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA117.000682

    β1,4GalTV regulates gliomagenesis and the transdifferentiation of glioma stem-like cells into endothelial cells depending on its galactosylation activity. A, total lysates of T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G) were analyzed with an RCA-1 lectin blot. The protein expression of GAPDH served as a loading control. B, nude mice were injected with T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). Four weeks later, photos were taken for the xenograft. Scale bars = 2 mm. C, Kaplan–Meier survival curves of nude mice injected with T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). D, immunofluorescence analysis shows co-localization of the human endothelial cell marker CD31 and the tumor cell marker GFP in tumor xenografts formed by T698968 cells expressing GFP and LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). Scale bars = 10 μm. E, the relative vascular density derived from glioma stem-like cells was quantified. Results are expressed as mean ± S.E. (n = 3; *, p < 0.05; **, p < 0.01). F, representative images of tube formation derived from T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). Scale bars = 20 μm. G, quantification of mean tube length in F. The results are expressed as mean ± S.E. (n = 3; ***, p < 0.001).
    Figure Legend Snippet: β1,4GalTV regulates gliomagenesis and the transdifferentiation of glioma stem-like cells into endothelial cells depending on its galactosylation activity. A, total lysates of T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G) were analyzed with an RCA-1 lectin blot. The protein expression of GAPDH served as a loading control. B, nude mice were injected with T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). Four weeks later, photos were taken for the xenograft. Scale bars = 2 mm. C, Kaplan–Meier survival curves of nude mice injected with T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). D, immunofluorescence analysis shows co-localization of the human endothelial cell marker CD31 and the tumor cell marker GFP in tumor xenografts formed by T698968 cells expressing GFP and LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). Scale bars = 10 μm. E, the relative vascular density derived from glioma stem-like cells was quantified. Results are expressed as mean ± S.E. (n = 3; *, p < 0.05; **, p < 0.01). F, representative images of tube formation derived from T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). Scale bars = 20 μm. G, quantification of mean tube length in F. The results are expressed as mean ± S.E. (n = 3; ***, p < 0.001).

    Techniques Used: Activity Assay, Expressing, shRNA, Injection, Immunofluorescence, Marker, Derivative Assay

    β1,4GalTV regulates the activity of Notch1 signaling. A, total lysates of T698968 cells expressing control or β1,4GalTV shRNA3 were analyzed with Notch1 antibody by Western blotting. The protein expression of GAPDH served as a loading control. B, the relative densities of NICD protein levels in A were quantified using densitometry. Values are normalized to that of T698968 cells expressing LacZ shRNA. Results are expressed as mean ± S.D. (n = 3; *, p < 0.05). C, Western blot analysis of nuclear NICD expression in T698968 cells expressing control or β1,4GalTV shRNA3. Sp1 served as a nuclear marker. D, RT-PCR analysis of Hes-1 expression in T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). E, the relative densities of Hes-1 PCR product levels were quantified using densitometry. Values are normalized to that of cells expressing LacZ shRNA + FLAG. Results are expressed as mean ± S.D. (n = 3; ***, p < 0.001). F, RT-PCR analysis of Notch1 expression in T698968 cells expressing control or β1,4GalTV shRNA. Actin expression served as a loading control. The relative densities of Notch1 PCR product levels were quantified using densitometry. Values are normalized to that of cells expressing control shRNA. **, p < 0.01. G, Western blot analysis of Notch1 from lysates of T698968 cells expressing control or β1,4GalTV shRNA in the absence or presence of the lysosome inhibitor chloroquine overnight. H, Notch1 proteins immunoprecipitated from T698968 cells expressing control or β1,4GalTV shRNA were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was overlaid with Notch1 antibody to verify the immunoprecipitation efficiency, with lectin RCA-1 to confirm β1,4-linked galactosylation, and with β1,4GalTV antibody to examine the interaction between β1,4GalTV and Notch1. IB, immunoblot. I, trafficking of newly synthesized Notch1 to the cell surface in T698968 cells expressing LacZ shRNA or β1,4GalTV shRNA3. Pulse labeling was performed with 1 mm AHA (a structural analog of methionine). The AHA-labeled Notch1 trafficked to the cell surface was examined using Western blotting. Results are expressed as percent of AHA-labeled Notch1 in cells expressing LacZ shRNA at 60 min. Data are represented as the means ± S.D. from three separate experiments. J, Notch1 proteins immunoprecipitated from T698968 cells expressing control or β1,4GalTV shRNA treated with His-tagged galectin-3 for 30 min were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was overlaid with Notch1 antibody to verify the IP efficiency and with His antibody to examine the interaction between His-tagged galectin-3 and Notch1. K, total lysates of T698968 cells expressing control or β1,4GalTV shRNA3 treated with or without His-tagged galectin-3 protein for 24 h were analyzed with Notch1 antibody by Western blotting. The protein expression of GAPDH served as a loading control. FL-Notch1, full-length Notch1.
    Figure Legend Snippet: β1,4GalTV regulates the activity of Notch1 signaling. A, total lysates of T698968 cells expressing control or β1,4GalTV shRNA3 were analyzed with Notch1 antibody by Western blotting. The protein expression of GAPDH served as a loading control. B, the relative densities of NICD protein levels in A were quantified using densitometry. Values are normalized to that of T698968 cells expressing LacZ shRNA. Results are expressed as mean ± S.D. (n = 3; *, p < 0.05). C, Western blot analysis of nuclear NICD expression in T698968 cells expressing control or β1,4GalTV shRNA3. Sp1 served as a nuclear marker. D, RT-PCR analysis of Hes-1 expression in T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). E, the relative densities of Hes-1 PCR product levels were quantified using densitometry. Values are normalized to that of cells expressing LacZ shRNA + FLAG. Results are expressed as mean ± S.D. (n = 3; ***, p < 0.001). F, RT-PCR analysis of Notch1 expression in T698968 cells expressing control or β1,4GalTV shRNA. Actin expression served as a loading control. The relative densities of Notch1 PCR product levels were quantified using densitometry. Values are normalized to that of cells expressing control shRNA. **, p < 0.01. G, Western blot analysis of Notch1 from lysates of T698968 cells expressing control or β1,4GalTV shRNA in the absence or presence of the lysosome inhibitor chloroquine overnight. H, Notch1 proteins immunoprecipitated from T698968 cells expressing control or β1,4GalTV shRNA were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was overlaid with Notch1 antibody to verify the immunoprecipitation efficiency, with lectin RCA-1 to confirm β1,4-linked galactosylation, and with β1,4GalTV antibody to examine the interaction between β1,4GalTV and Notch1. IB, immunoblot. I, trafficking of newly synthesized Notch1 to the cell surface in T698968 cells expressing LacZ shRNA or β1,4GalTV shRNA3. Pulse labeling was performed with 1 mm AHA (a structural analog of methionine). The AHA-labeled Notch1 trafficked to the cell surface was examined using Western blotting. Results are expressed as percent of AHA-labeled Notch1 in cells expressing LacZ shRNA at 60 min. Data are represented as the means ± S.D. from three separate experiments. J, Notch1 proteins immunoprecipitated from T698968 cells expressing control or β1,4GalTV shRNA treated with His-tagged galectin-3 for 30 min were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was overlaid with Notch1 antibody to verify the IP efficiency and with His antibody to examine the interaction between His-tagged galectin-3 and Notch1. K, total lysates of T698968 cells expressing control or β1,4GalTV shRNA3 treated with or without His-tagged galectin-3 protein for 24 h were analyzed with Notch1 antibody by Western blotting. The protein expression of GAPDH served as a loading control. FL-Notch1, full-length Notch1.

    Techniques Used: Activity Assay, Expressing, Western Blot, shRNA, Marker, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, SDS Page, Synthesized, Labeling

    biotinylated lectin ricinus communis agglutinin i rca 1  (Vector Laboratories)


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    Vector Laboratories biotinylated lectin ricinus communis agglutinin i rca 1
    β1,4GalTV regulates gliomagenesis and the transdifferentiation of glioma stem-like cells into endothelial cells depending on its galactosylation activity. A, total lysates of T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G) were analyzed with an <t>RCA-1</t> lectin blot. The protein expression of GAPDH served as a loading control. B, nude mice were injected with T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). Four weeks later, photos were taken for the xenograft. Scale bars = 2 mm. C, Kaplan–Meier survival curves of nude mice injected with T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). D, immunofluorescence analysis shows co-localization of the human endothelial cell marker CD31 and the tumor cell marker GFP in tumor xenografts formed by T698968 cells expressing GFP and LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). Scale bars = 10 μm. E, the relative vascular density derived from glioma stem-like cells was quantified. Results are expressed as mean ± S.E. (n = 3; *, p < 0.05; **, p < 0.01). F, representative images of tube formation derived from T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). Scale bars = 20 μm. G, quantification of mean tube length in F. The results are expressed as mean ± S.E. (n = 3; ***, p < 0.001).
    Biotinylated Lectin Ricinus Communis Agglutinin I Rca 1, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "β1,4-Galactosyltransferase V activates Notch1 signaling in glioma stem-like cells and promotes their transdifferentiation into endothelial cells"

    Article Title: β1,4-Galactosyltransferase V activates Notch1 signaling in glioma stem-like cells and promotes their transdifferentiation into endothelial cells

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA117.000682

    β1,4GalTV regulates gliomagenesis and the transdifferentiation of glioma stem-like cells into endothelial cells depending on its galactosylation activity. A, total lysates of T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G) were analyzed with an RCA-1 lectin blot. The protein expression of GAPDH served as a loading control. B, nude mice were injected with T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). Four weeks later, photos were taken for the xenograft. Scale bars = 2 mm. C, Kaplan–Meier survival curves of nude mice injected with T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). D, immunofluorescence analysis shows co-localization of the human endothelial cell marker CD31 and the tumor cell marker GFP in tumor xenografts formed by T698968 cells expressing GFP and LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). Scale bars = 10 μm. E, the relative vascular density derived from glioma stem-like cells was quantified. Results are expressed as mean ± S.E. (n = 3; *, p < 0.05; **, p < 0.01). F, representative images of tube formation derived from T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). Scale bars = 20 μm. G, quantification of mean tube length in F. The results are expressed as mean ± S.E. (n = 3; ***, p < 0.001).
    Figure Legend Snippet: β1,4GalTV regulates gliomagenesis and the transdifferentiation of glioma stem-like cells into endothelial cells depending on its galactosylation activity. A, total lysates of T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G) were analyzed with an RCA-1 lectin blot. The protein expression of GAPDH served as a loading control. B, nude mice were injected with T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). Four weeks later, photos were taken for the xenograft. Scale bars = 2 mm. C, Kaplan–Meier survival curves of nude mice injected with T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). D, immunofluorescence analysis shows co-localization of the human endothelial cell marker CD31 and the tumor cell marker GFP in tumor xenografts formed by T698968 cells expressing GFP and LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). Scale bars = 10 μm. E, the relative vascular density derived from glioma stem-like cells was quantified. Results are expressed as mean ± S.E. (n = 3; *, p < 0.05; **, p < 0.01). F, representative images of tube formation derived from T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). Scale bars = 20 μm. G, quantification of mean tube length in F. The results are expressed as mean ± S.E. (n = 3; ***, p < 0.001).

    Techniques Used: Activity Assay, Expressing, shRNA, Injection, Immunofluorescence, Marker, Derivative Assay

    β1,4GalTV regulates the activity of Notch1 signaling. A, total lysates of T698968 cells expressing control or β1,4GalTV shRNA3 were analyzed with Notch1 antibody by Western blotting. The protein expression of GAPDH served as a loading control. B, the relative densities of NICD protein levels in A were quantified using densitometry. Values are normalized to that of T698968 cells expressing LacZ shRNA. Results are expressed as mean ± S.D. (n = 3; *, p < 0.05). C, Western blot analysis of nuclear NICD expression in T698968 cells expressing control or β1,4GalTV shRNA3. Sp1 served as a nuclear marker. D, RT-PCR analysis of Hes-1 expression in T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). E, the relative densities of Hes-1 PCR product levels were quantified using densitometry. Values are normalized to that of cells expressing LacZ shRNA + FLAG. Results are expressed as mean ± S.D. (n = 3; ***, p < 0.001). F, RT-PCR analysis of Notch1 expression in T698968 cells expressing control or β1,4GalTV shRNA. Actin expression served as a loading control. The relative densities of Notch1 PCR product levels were quantified using densitometry. Values are normalized to that of cells expressing control shRNA. **, p < 0.01. G, Western blot analysis of Notch1 from lysates of T698968 cells expressing control or β1,4GalTV shRNA in the absence or presence of the lysosome inhibitor chloroquine overnight. H, Notch1 proteins immunoprecipitated from T698968 cells expressing control or β1,4GalTV shRNA were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was overlaid with Notch1 antibody to verify the immunoprecipitation efficiency, with lectin RCA-1 to confirm β1,4-linked galactosylation, and with β1,4GalTV antibody to examine the interaction between β1,4GalTV and Notch1. IB, immunoblot. I, trafficking of newly synthesized Notch1 to the cell surface in T698968 cells expressing LacZ shRNA or β1,4GalTV shRNA3. Pulse labeling was performed with 1 mm AHA (a structural analog of methionine). The AHA-labeled Notch1 trafficked to the cell surface was examined using Western blotting. Results are expressed as percent of AHA-labeled Notch1 in cells expressing LacZ shRNA at 60 min. Data are represented as the means ± S.D. from three separate experiments. J, Notch1 proteins immunoprecipitated from T698968 cells expressing control or β1,4GalTV shRNA treated with His-tagged galectin-3 for 30 min were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was overlaid with Notch1 antibody to verify the IP efficiency and with His antibody to examine the interaction between His-tagged galectin-3 and Notch1. K, total lysates of T698968 cells expressing control or β1,4GalTV shRNA3 treated with or without His-tagged galectin-3 protein for 24 h were analyzed with Notch1 antibody by Western blotting. The protein expression of GAPDH served as a loading control. FL-Notch1, full-length Notch1.
    Figure Legend Snippet: β1,4GalTV regulates the activity of Notch1 signaling. A, total lysates of T698968 cells expressing control or β1,4GalTV shRNA3 were analyzed with Notch1 antibody by Western blotting. The protein expression of GAPDH served as a loading control. B, the relative densities of NICD protein levels in A were quantified using densitometry. Values are normalized to that of T698968 cells expressing LacZ shRNA. Results are expressed as mean ± S.D. (n = 3; *, p < 0.05). C, Western blot analysis of nuclear NICD expression in T698968 cells expressing control or β1,4GalTV shRNA3. Sp1 served as a nuclear marker. D, RT-PCR analysis of Hes-1 expression in T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). E, the relative densities of Hes-1 PCR product levels were quantified using densitometry. Values are normalized to that of cells expressing LacZ shRNA + FLAG. Results are expressed as mean ± S.D. (n = 3; ***, p < 0.001). F, RT-PCR analysis of Notch1 expression in T698968 cells expressing control or β1,4GalTV shRNA. Actin expression served as a loading control. The relative densities of Notch1 PCR product levels were quantified using densitometry. Values are normalized to that of cells expressing control shRNA. **, p < 0.01. G, Western blot analysis of Notch1 from lysates of T698968 cells expressing control or β1,4GalTV shRNA in the absence or presence of the lysosome inhibitor chloroquine overnight. H, Notch1 proteins immunoprecipitated from T698968 cells expressing control or β1,4GalTV shRNA were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was overlaid with Notch1 antibody to verify the immunoprecipitation efficiency, with lectin RCA-1 to confirm β1,4-linked galactosylation, and with β1,4GalTV antibody to examine the interaction between β1,4GalTV and Notch1. IB, immunoblot. I, trafficking of newly synthesized Notch1 to the cell surface in T698968 cells expressing LacZ shRNA or β1,4GalTV shRNA3. Pulse labeling was performed with 1 mm AHA (a structural analog of methionine). The AHA-labeled Notch1 trafficked to the cell surface was examined using Western blotting. Results are expressed as percent of AHA-labeled Notch1 in cells expressing LacZ shRNA at 60 min. Data are represented as the means ± S.D. from three separate experiments. J, Notch1 proteins immunoprecipitated from T698968 cells expressing control or β1,4GalTV shRNA treated with His-tagged galectin-3 for 30 min were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was overlaid with Notch1 antibody to verify the IP efficiency and with His antibody to examine the interaction between His-tagged galectin-3 and Notch1. K, total lysates of T698968 cells expressing control or β1,4GalTV shRNA3 treated with or without His-tagged galectin-3 protein for 24 h were analyzed with Notch1 antibody by Western blotting. The protein expression of GAPDH served as a loading control. FL-Notch1, full-length Notch1.

    Techniques Used: Activity Assay, Expressing, Western Blot, shRNA, Marker, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, SDS Page, Synthesized, Labeling

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    Vector Laboratories lectin ricinus communis agglutinin 1 rca 1
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    Immunohistochemical illustration of Ricinus communis <t>agglutinin-1</t> <t>(RCA-1)</t> -positive microglial cells in the white matter of spinal cord . The RCA-1-positive microglial cells (arrowheads) in both healthy (A) and AvIDP-affected (B) chicken show incospicuous ramified microglial cells. Activated microglial cells or an increased number were not observed. A, B: asterisk = endothelium (also stains RCA-1 positive); scale bar = 250 μm.
    Biotinylated Lectin Ricinus Communis Agglutinin 1, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Immunohistochemical illustration of Ricinus communis <t>agglutinin-1</t> <t>(RCA-1)</t> -positive microglial cells in the white matter of spinal cord . The RCA-1-positive microglial cells (arrowheads) in both healthy (A) and AvIDP-affected (B) chicken show incospicuous ramified microglial cells. Activated microglial cells or an increased number were not observed. A, B: asterisk = endothelium (also stains RCA-1 positive); scale bar = 250 μm.
    Ricinus Communis Agglutinin I Rca 1 Lectins, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Vector Laboratories lectin ricinus communis agglutinin 1
    Graphs represent the qualitative analysis (cells/mm 2 ) of activated microglia (( A <t>)—RCA-1</t> positive microglia; ( B )—Mac-3 positive microglia, ( C )—proliferating RCA-1/Ki-67 positive microglia), and GFAP positive astrocytes ( D ) during early remyelination (week 5.5) in the corpus callosum of animals treated with different dosages of CDP-choline or sham (PBS). The astrogliosis was not affected by CDP-choline treatment. However, the number of activated microglia in all animals treated with CDP-choline, regardless of the dose used, was significantly lower than in the control group. Significant effects between mice treated with different dosages of CDP-choline and PBS are indicated by asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001, n = 6).
    Lectin Ricinus Communis Agglutinin 1, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Vector Laboratories rhodamine labeled ricinus communis agglutinin i (rca i, rca120)

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    Vector Laboratories ricinus communis agglutinin 1 rca 1 lectin
    NLRP12 inhibition regulates cellular proliferation. ( a ) NLRP12 expression was observed in glioma (LN18) and microglial (BV2) cells using immunofluorescence (magnification: 40X, scale bar: 50 μm). ( b ) Colony formation assay shows increased cellular proliferation in the NLRP12si treated microglia. Magnification: 40X. ( c , d ) Quantification of both BV2 and LN18 cells per colony. ( e ) Effect of LN18 (NLRP12 siRNA treated) cell conditioned medium (CM) on BV2 cell migration was assessed using migration assay. 8 image sections per sample were quantified for migration analysis. Student’s t-test and one-way AVOVA were performed to find the statistical significance of colony formation and migration assay respectively (p-value; *<0.05, **<0.005). The error bar indicates standard error of mean. ( f ) NLRP12 expression in human normal brain tissue and glioma sections, using anti-NLRP12 antibody (N = 7). Nuclei were stained blue with DAPI and microglia were stained green with FITC tagged lectin RCA (Ricinus Communis <t>Agglutinin-1);</t> Magnification: 20X, scale bar: 100 µm.
    Ricinus Communis Agglutinin 1 Rca 1 Lectin, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    β1,4GalTV regulates gliomagenesis and the transdifferentiation of glioma stem-like cells into endothelial cells depending on its galactosylation activity. A, total lysates of T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G) were analyzed with an <t>RCA-1</t> lectin blot. The protein expression of GAPDH served as a loading control. B, nude mice were injected with T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). Four weeks later, photos were taken for the xenograft. Scale bars = 2 mm. C, Kaplan–Meier survival curves of nude mice injected with T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). D, immunofluorescence analysis shows co-localization of the human endothelial cell marker CD31 and the tumor cell marker GFP in tumor xenografts formed by T698968 cells expressing GFP and LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). Scale bars = 10 μm. E, the relative vascular density derived from glioma stem-like cells was quantified. Results are expressed as mean ± S.E. (n = 3; *, p < 0.05; **, p < 0.01). F, representative images of tube formation derived from T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). Scale bars = 20 μm. G, quantification of mean tube length in F. The results are expressed as mean ± S.E. (n = 3; ***, p < 0.001).
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    Immunohistochemical illustration of Ricinus communis agglutinin-1 (RCA-1) -positive microglial cells in the white matter of spinal cord . The RCA-1-positive microglial cells (arrowheads) in both healthy (A) and AvIDP-affected (B) chicken show incospicuous ramified microglial cells. Activated microglial cells or an increased number were not observed. A, B: asterisk = endothelium (also stains RCA-1 positive); scale bar = 250 μm.

    Journal: Journal of Neuroinflammation

    Article Title: Acute paretic syndrome in juvenile White Leghorn chickens resembles late stages of acute inflammatory demyelinating polyneuropathies in humans

    doi: 10.1186/1742-2094-7-7

    Figure Lengend Snippet: Immunohistochemical illustration of Ricinus communis agglutinin-1 (RCA-1) -positive microglial cells in the white matter of spinal cord . The RCA-1-positive microglial cells (arrowheads) in both healthy (A) and AvIDP-affected (B) chicken show incospicuous ramified microglial cells. Activated microglial cells or an increased number were not observed. A, B: asterisk = endothelium (also stains RCA-1 positive); scale bar = 250 μm.

    Article Snippet: Furthermore, the biotinylated lectin Ricinus communis agglutinin-1 (RCA 120, 1:3000; Vector Laboratories, CA, USA) was used as a specific marker for microglial cells within the spinal cord [ ].

    Techniques: Immunohistochemical staining

    Graphs represent the qualitative analysis (cells/mm 2 ) of activated microglia (( A )—RCA-1 positive microglia; ( B )—Mac-3 positive microglia, ( C )—proliferating RCA-1/Ki-67 positive microglia), and GFAP positive astrocytes ( D ) during early remyelination (week 5.5) in the corpus callosum of animals treated with different dosages of CDP-choline or sham (PBS). The astrogliosis was not affected by CDP-choline treatment. However, the number of activated microglia in all animals treated with CDP-choline, regardless of the dose used, was significantly lower than in the control group. Significant effects between mice treated with different dosages of CDP-choline and PBS are indicated by asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001, n = 6).

    Journal: Pharmaceuticals

    Article Title: Regenerative Effects of CDP-Choline: A Dose-Dependent Study in the Toxic Cuprizone Model of De- and Remyelination

    doi: 10.3390/ph14111156

    Figure Lengend Snippet: Graphs represent the qualitative analysis (cells/mm 2 ) of activated microglia (( A )—RCA-1 positive microglia; ( B )—Mac-3 positive microglia, ( C )—proliferating RCA-1/Ki-67 positive microglia), and GFAP positive astrocytes ( D ) during early remyelination (week 5.5) in the corpus callosum of animals treated with different dosages of CDP-choline or sham (PBS). The astrogliosis was not affected by CDP-choline treatment. However, the number of activated microglia in all animals treated with CDP-choline, regardless of the dose used, was significantly lower than in the control group. Significant effects between mice treated with different dosages of CDP-choline and PBS are indicated by asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001, n = 6).

    Article Snippet: The following primary antibodies were used: for myelin proteolipid protein (PLP) (mouse monoclonal IgG2a, 1:500, Serotec) and myelin basic protein (MBP) (mouse monoclonal IgG2b, 1:500, Covance), for activated microglia Mac-3 (rat IgG, 1:500, BD Pharmingen) and the lectin ricinus communis agglutinin 1 (RCA-1) (1:1000, biotinylated, Vector Laboratories), for astrocytes glial fibrillary acidic protein (GFAP) (mouse IgG, 1:200, Millipore), for oligodendrocytes Nogo-A (rabbit IgG, 1:750, Millipore) and anti-adenomatus polyposis coli (APC; mouse IgG; 1:200).

    Techniques:

    Journal: STAR Protocols

    Article Title: Protocol for induction and characterization of microglia extracellular traps in murine and human microglia cells

    doi: 10.1016/j.xpro.2021.100678

    Figure Lengend Snippet:

    Article Snippet: Fluorescein Ricinus communis agglutinin-1 lectin , Vector Labs , FL-1081.

    Techniques: Recombinant, Staining, Plasmid Preparation, Software, Phagocytosis Assay, Microscopy

    NLRP12 inhibition regulates cellular proliferation. ( a ) NLRP12 expression was observed in glioma (LN18) and microglial (BV2) cells using immunofluorescence (magnification: 40X, scale bar: 50 μm). ( b ) Colony formation assay shows increased cellular proliferation in the NLRP12si treated microglia. Magnification: 40X. ( c , d ) Quantification of both BV2 and LN18 cells per colony. ( e ) Effect of LN18 (NLRP12 siRNA treated) cell conditioned medium (CM) on BV2 cell migration was assessed using migration assay. 8 image sections per sample were quantified for migration analysis. Student’s t-test and one-way AVOVA were performed to find the statistical significance of colony formation and migration assay respectively (p-value; *<0.05, **<0.005). The error bar indicates standard error of mean. ( f ) NLRP12 expression in human normal brain tissue and glioma sections, using anti-NLRP12 antibody (N = 7). Nuclei were stained blue with DAPI and microglia were stained green with FITC tagged lectin RCA (Ricinus Communis Agglutinin-1); Magnification: 20X, scale bar: 100 µm.

    Journal: Scientific Reports

    Article Title: Differential Expression Profile of NLRs and AIM2 in Glioma and Implications for NLRP12 in Glioblastoma

    doi: 10.1038/s41598-019-44854-4

    Figure Lengend Snippet: NLRP12 inhibition regulates cellular proliferation. ( a ) NLRP12 expression was observed in glioma (LN18) and microglial (BV2) cells using immunofluorescence (magnification: 40X, scale bar: 50 μm). ( b ) Colony formation assay shows increased cellular proliferation in the NLRP12si treated microglia. Magnification: 40X. ( c , d ) Quantification of both BV2 and LN18 cells per colony. ( e ) Effect of LN18 (NLRP12 siRNA treated) cell conditioned medium (CM) on BV2 cell migration was assessed using migration assay. 8 image sections per sample were quantified for migration analysis. Student’s t-test and one-way AVOVA were performed to find the statistical significance of colony formation and migration assay respectively (p-value; *<0.05, **<0.005). The error bar indicates standard error of mean. ( f ) NLRP12 expression in human normal brain tissue and glioma sections, using anti-NLRP12 antibody (N = 7). Nuclei were stained blue with DAPI and microglia were stained green with FITC tagged lectin RCA (Ricinus Communis Agglutinin-1); Magnification: 20X, scale bar: 100 µm.

    Article Snippet: For the detection of microglia, tissues were stained with Ricinus communis agglutinin-1 (RCA-1) lectin (Vector labs, FL-1081) .

    Techniques: Inhibition, Expressing, Immunofluorescence, Colony Assay, Migration, Staining

    β1,4GalTV regulates gliomagenesis and the transdifferentiation of glioma stem-like cells into endothelial cells depending on its galactosylation activity. A, total lysates of T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G) were analyzed with an RCA-1 lectin blot. The protein expression of GAPDH served as a loading control. B, nude mice were injected with T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). Four weeks later, photos were taken for the xenograft. Scale bars = 2 mm. C, Kaplan–Meier survival curves of nude mice injected with T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). D, immunofluorescence analysis shows co-localization of the human endothelial cell marker CD31 and the tumor cell marker GFP in tumor xenografts formed by T698968 cells expressing GFP and LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). Scale bars = 10 μm. E, the relative vascular density derived from glioma stem-like cells was quantified. Results are expressed as mean ± S.E. (n = 3; *, p < 0.05; **, p < 0.01). F, representative images of tube formation derived from T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). Scale bars = 20 μm. G, quantification of mean tube length in F. The results are expressed as mean ± S.E. (n = 3; ***, p < 0.001).

    Journal: The Journal of Biological Chemistry

    Article Title: β1,4-Galactosyltransferase V activates Notch1 signaling in glioma stem-like cells and promotes their transdifferentiation into endothelial cells

    doi: 10.1074/jbc.RA117.000682

    Figure Lengend Snippet: β1,4GalTV regulates gliomagenesis and the transdifferentiation of glioma stem-like cells into endothelial cells depending on its galactosylation activity. A, total lysates of T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G) were analyzed with an RCA-1 lectin blot. The protein expression of GAPDH served as a loading control. B, nude mice were injected with T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). Four weeks later, photos were taken for the xenograft. Scale bars = 2 mm. C, Kaplan–Meier survival curves of nude mice injected with T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). D, immunofluorescence analysis shows co-localization of the human endothelial cell marker CD31 and the tumor cell marker GFP in tumor xenografts formed by T698968 cells expressing GFP and LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). Scale bars = 10 μm. E, the relative vascular density derived from glioma stem-like cells was quantified. Results are expressed as mean ± S.E. (n = 3; *, p < 0.05; **, p < 0.01). F, representative images of tube formation derived from T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). Scale bars = 20 μm. G, quantification of mean tube length in F. The results are expressed as mean ± S.E. (n = 3; ***, p < 0.001).

    Article Snippet: The primary antibody was biotinylated lectin Ricinus communis agglutinin I (RCA-1) (Vector, catalog no. B-1085).

    Techniques: Activity Assay, Expressing, shRNA, Injection, Immunofluorescence, Marker, Derivative Assay

    β1,4GalTV regulates the activity of Notch1 signaling. A, total lysates of T698968 cells expressing control or β1,4GalTV shRNA3 were analyzed with Notch1 antibody by Western blotting. The protein expression of GAPDH served as a loading control. B, the relative densities of NICD protein levels in A were quantified using densitometry. Values are normalized to that of T698968 cells expressing LacZ shRNA. Results are expressed as mean ± S.D. (n = 3; *, p < 0.05). C, Western blot analysis of nuclear NICD expression in T698968 cells expressing control or β1,4GalTV shRNA3. Sp1 served as a nuclear marker. D, RT-PCR analysis of Hes-1 expression in T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). E, the relative densities of Hes-1 PCR product levels were quantified using densitometry. Values are normalized to that of cells expressing LacZ shRNA + FLAG. Results are expressed as mean ± S.D. (n = 3; ***, p < 0.001). F, RT-PCR analysis of Notch1 expression in T698968 cells expressing control or β1,4GalTV shRNA. Actin expression served as a loading control. The relative densities of Notch1 PCR product levels were quantified using densitometry. Values are normalized to that of cells expressing control shRNA. **, p < 0.01. G, Western blot analysis of Notch1 from lysates of T698968 cells expressing control or β1,4GalTV shRNA in the absence or presence of the lysosome inhibitor chloroquine overnight. H, Notch1 proteins immunoprecipitated from T698968 cells expressing control or β1,4GalTV shRNA were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was overlaid with Notch1 antibody to verify the immunoprecipitation efficiency, with lectin RCA-1 to confirm β1,4-linked galactosylation, and with β1,4GalTV antibody to examine the interaction between β1,4GalTV and Notch1. IB, immunoblot. I, trafficking of newly synthesized Notch1 to the cell surface in T698968 cells expressing LacZ shRNA or β1,4GalTV shRNA3. Pulse labeling was performed with 1 mm AHA (a structural analog of methionine). The AHA-labeled Notch1 trafficked to the cell surface was examined using Western blotting. Results are expressed as percent of AHA-labeled Notch1 in cells expressing LacZ shRNA at 60 min. Data are represented as the means ± S.D. from three separate experiments. J, Notch1 proteins immunoprecipitated from T698968 cells expressing control or β1,4GalTV shRNA treated with His-tagged galectin-3 for 30 min were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was overlaid with Notch1 antibody to verify the IP efficiency and with His antibody to examine the interaction between His-tagged galectin-3 and Notch1. K, total lysates of T698968 cells expressing control or β1,4GalTV shRNA3 treated with or without His-tagged galectin-3 protein for 24 h were analyzed with Notch1 antibody by Western blotting. The protein expression of GAPDH served as a loading control. FL-Notch1, full-length Notch1.

    Journal: The Journal of Biological Chemistry

    Article Title: β1,4-Galactosyltransferase V activates Notch1 signaling in glioma stem-like cells and promotes their transdifferentiation into endothelial cells

    doi: 10.1074/jbc.RA117.000682

    Figure Lengend Snippet: β1,4GalTV regulates the activity of Notch1 signaling. A, total lysates of T698968 cells expressing control or β1,4GalTV shRNA3 were analyzed with Notch1 antibody by Western blotting. The protein expression of GAPDH served as a loading control. B, the relative densities of NICD protein levels in A were quantified using densitometry. Values are normalized to that of T698968 cells expressing LacZ shRNA. Results are expressed as mean ± S.D. (n = 3; *, p < 0.05). C, Western blot analysis of nuclear NICD expression in T698968 cells expressing control or β1,4GalTV shRNA3. Sp1 served as a nuclear marker. D, RT-PCR analysis of Hes-1 expression in T698968 cells expressing LacZ shRNA + FLAG, β1,4GalTV shRNA3 + FLAG, β1,4GalTV shRNA3 + GalTV, or β1,4GalTV shRNA3 + GalTV (Y268G/W294G). E, the relative densities of Hes-1 PCR product levels were quantified using densitometry. Values are normalized to that of cells expressing LacZ shRNA + FLAG. Results are expressed as mean ± S.D. (n = 3; ***, p < 0.001). F, RT-PCR analysis of Notch1 expression in T698968 cells expressing control or β1,4GalTV shRNA. Actin expression served as a loading control. The relative densities of Notch1 PCR product levels were quantified using densitometry. Values are normalized to that of cells expressing control shRNA. **, p < 0.01. G, Western blot analysis of Notch1 from lysates of T698968 cells expressing control or β1,4GalTV shRNA in the absence or presence of the lysosome inhibitor chloroquine overnight. H, Notch1 proteins immunoprecipitated from T698968 cells expressing control or β1,4GalTV shRNA were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was overlaid with Notch1 antibody to verify the immunoprecipitation efficiency, with lectin RCA-1 to confirm β1,4-linked galactosylation, and with β1,4GalTV antibody to examine the interaction between β1,4GalTV and Notch1. IB, immunoblot. I, trafficking of newly synthesized Notch1 to the cell surface in T698968 cells expressing LacZ shRNA or β1,4GalTV shRNA3. Pulse labeling was performed with 1 mm AHA (a structural analog of methionine). The AHA-labeled Notch1 trafficked to the cell surface was examined using Western blotting. Results are expressed as percent of AHA-labeled Notch1 in cells expressing LacZ shRNA at 60 min. Data are represented as the means ± S.D. from three separate experiments. J, Notch1 proteins immunoprecipitated from T698968 cells expressing control or β1,4GalTV shRNA treated with His-tagged galectin-3 for 30 min were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was overlaid with Notch1 antibody to verify the IP efficiency and with His antibody to examine the interaction between His-tagged galectin-3 and Notch1. K, total lysates of T698968 cells expressing control or β1,4GalTV shRNA3 treated with or without His-tagged galectin-3 protein for 24 h were analyzed with Notch1 antibody by Western blotting. The protein expression of GAPDH served as a loading control. FL-Notch1, full-length Notch1.

    Article Snippet: The primary antibody was biotinylated lectin Ricinus communis agglutinin I (RCA-1) (Vector, catalog no. B-1085).

    Techniques: Activity Assay, Expressing, Western Blot, shRNA, Marker, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, SDS Page, Synthesized, Labeling