fucosidase  (New England Biolabs)


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  • 95
    Name:
    alpha 1 2 Fucosidase
    Description:
    alpha 1 2 Fucosidase 5 000 units
    Catalog Number:
    P0724L
    Price:
    520
    Category:
    Glycosidases
    Size:
    5 000 units
    Buy from Supplier


    Structured Review

    New England Biolabs fucosidase
    alpha 1 2 Fucosidase
    alpha 1 2 Fucosidase 5 000 units
    https://www.bioz.com/result/fucosidase/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    fucosidase - by Bioz Stars, 2021-06
    95/100 stars

    Images

    1) Product Images from "Human stem cell-derived retinal epithelial cells activate complement via collectin 11 in response to stress"

    Article Title: Human stem cell-derived retinal epithelial cells activate complement via collectin 11 in response to stress

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-15212-z

    C3d expression and MAC formation on hypoxia-stressed iPS-RPE cells following fucosidase treatment. ( a ) Representative immunofluorescence images showing C3d deposition on the surface of hypoxia-stressed iPS-RPE cells treated or not overnight with fucosidase. C3d (green), nuclei nuclei (blue) and phalloidin (grey) are shown. Scale bars, 50 μm. ( b ) Quantification of C3d expression level on hypoxia-stressed iPS- RPE cells compared to fucosidase-treated hypoxic cells using ImageJ software. ( c ) Representative immunofluorescence images showing MAC staining on the surface of hypoxia-stressed iPS-RPE cells treated or not overnight with fucosidase. MAC (red), nuclei (blue) and phalloidin (grey) are shown. Scale bars, 50 μm.
    Figure Legend Snippet: C3d expression and MAC formation on hypoxia-stressed iPS-RPE cells following fucosidase treatment. ( a ) Representative immunofluorescence images showing C3d deposition on the surface of hypoxia-stressed iPS-RPE cells treated or not overnight with fucosidase. C3d (green), nuclei nuclei (blue) and phalloidin (grey) are shown. Scale bars, 50 μm. ( b ) Quantification of C3d expression level on hypoxia-stressed iPS- RPE cells compared to fucosidase-treated hypoxic cells using ImageJ software. ( c ) Representative immunofluorescence images showing MAC staining on the surface of hypoxia-stressed iPS-RPE cells treated or not overnight with fucosidase. MAC (red), nuclei (blue) and phalloidin (grey) are shown. Scale bars, 50 μm.

    Techniques Used: Expressing, Immunofluorescence, Software, Staining

    2) Product Images from "Toward robust N-glycomics of various tissue samples that may contain glycans with unknown or unexpected structures"

    Article Title: Toward robust N-glycomics of various tissue samples that may contain glycans with unknown or unexpected structures

    Journal: Scientific Reports

    doi: 10.1038/s41598-021-84668-x

    Distinct elution positions of PA- N -glycans on reversed-phase LC based on their different core structures and branching patterns. ( A ) EICs at m/z 860.33 [Hex 2 HexNAc 2 C-PA(2H + ), orange line], 933.36 [Hex 2 HexNAc 2 Fuc 1 C-PA(2H + ), black line], 961.87 [Hex 2 HexNAc 3 C-PA(2H + ), blue line], and 1034.90 [Hex 2 HexNAc 3 Fuc 1 C-PA(2H + ), red line] of biantennary PA- N -glycans in neuraminidase/α1-3,4 fucosidase–treated fr. 3 from chicken colon. ( B ) EICs at m/z 698.28 [HexNAc 2 C-PA(2H + ), orange line], 771.31 [HexNAc 2 Fuc 1 C-PA(2H + ), black line], 799.82 [HexNAc 3 C-PA(2H + ), blue line], and 872.85 [HexNAc 3 Fuc 1 C-PA(2H + ), red line] of biantennary PA- N -glycans in neuraminidase/α1-3,4 fucosidase/β1-4 galactosidase–treated fr. 3. ( C ) EICs at m/z 933.36, 1115.93, 866.00, 987.71, and 1109.42 of PA- N -glycans in neuraminidase/α1-3,4 fucosidase–treated fr. 3 or fr.4. The peaks indicated by an asterisk (*) are probably artificial ion signals derived from large amounts of PA- N -glycans eluted around the corresponding times. ( D ) EICs at m/z 771.31, 872.85, 974.39, 1075.93, and 1177.47 of PA- N -glycans in neuraminidase/α1-3,4 fucosidase/β1-4 galactosidase–treated fr. 1, 3 or 4.
    Figure Legend Snippet: Distinct elution positions of PA- N -glycans on reversed-phase LC based on their different core structures and branching patterns. ( A ) EICs at m/z 860.33 [Hex 2 HexNAc 2 C-PA(2H + ), orange line], 933.36 [Hex 2 HexNAc 2 Fuc 1 C-PA(2H + ), black line], 961.87 [Hex 2 HexNAc 3 C-PA(2H + ), blue line], and 1034.90 [Hex 2 HexNAc 3 Fuc 1 C-PA(2H + ), red line] of biantennary PA- N -glycans in neuraminidase/α1-3,4 fucosidase–treated fr. 3 from chicken colon. ( B ) EICs at m/z 698.28 [HexNAc 2 C-PA(2H + ), orange line], 771.31 [HexNAc 2 Fuc 1 C-PA(2H + ), black line], 799.82 [HexNAc 3 C-PA(2H + ), blue line], and 872.85 [HexNAc 3 Fuc 1 C-PA(2H + ), red line] of biantennary PA- N -glycans in neuraminidase/α1-3,4 fucosidase/β1-4 galactosidase–treated fr. 3. ( C ) EICs at m/z 933.36, 1115.93, 866.00, 987.71, and 1109.42 of PA- N -glycans in neuraminidase/α1-3,4 fucosidase–treated fr. 3 or fr.4. The peaks indicated by an asterisk (*) are probably artificial ion signals derived from large amounts of PA- N -glycans eluted around the corresponding times. ( D ) EICs at m/z 771.31, 872.85, 974.39, 1075.93, and 1177.47 of PA- N -glycans in neuraminidase/α1-3,4 fucosidase/β1-4 galactosidase–treated fr. 1, 3 or 4.

    Techniques Used: Derivative Assay

    Elution profiles of PA- N -glycans from chicken colon after sequential digestions with exoglycosidases. A portion of fr.3 from the DEAE column (monosialylated PA- N -glycans, see Supplementary Fig. S2 A) was sequentially digested with neuraminidase, α1-3,4 fucosidase, and β1-4 galactosidase, and each digest was subjected to LC–MS and MS/MS analysis. Arrows with alphabetical characters indicate the elution positions of the standard PA- N -glycans (Supplementary Fig. S1 ). Some representative PA- N -glycans, but not all the structures detected by MS/MS, are shown in this figure. The standard Symbol Nomenclature for Glycan system was used for monosaccharide symbols 51 , except for sulfate and phosphate groups.
    Figure Legend Snippet: Elution profiles of PA- N -glycans from chicken colon after sequential digestions with exoglycosidases. A portion of fr.3 from the DEAE column (monosialylated PA- N -glycans, see Supplementary Fig. S2 A) was sequentially digested with neuraminidase, α1-3,4 fucosidase, and β1-4 galactosidase, and each digest was subjected to LC–MS and MS/MS analysis. Arrows with alphabetical characters indicate the elution positions of the standard PA- N -glycans (Supplementary Fig. S1 ). Some representative PA- N -glycans, but not all the structures detected by MS/MS, are shown in this figure. The standard Symbol Nomenclature for Glycan system was used for monosaccharide symbols 51 , except for sulfate and phosphate groups.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy

    3) Product Images from "TSTA3 facilitates esophageal squamous cell carcinoma progression through regulating fucosylation of LAMP2 and ERBB2"

    Article Title: TSTA3 facilitates esophageal squamous cell carcinoma progression through regulating fucosylation of LAMP2 and ERBB2

    Journal: Theranostics

    doi: 10.7150/thno.48225

    Identification of fucosylated glycoproteins in ESCC revealed regulators of invasion and metastasis. (A) Schematic illustration of the experimental approach showing affinity enrichment of core-fucosylated and α-1,2-fucosylated proteins by LCA and UEA-I lectin affinity chromatography respectively. (B) The effect of enriched fucosylated protein by UEA-I lectin with or without α-1,2-fucosidase on KYSE150 and KYSE450 cell invasion. (C) Coomassie brilliant blue (CBB) staining of gels of whole cell lysate proteins and LCA enriched fucosylated proteins in TSTA3-WT and control group. (D) Number of overlapping proteins between lectin enriched proteins identified by in gel mass spectrometry analysis and differentially expressed glycoproteins in N-glycoproteomics data. (E) LCA affinity chromatography of whole-cell lysates of ESCC cells transfected with TSTA3-WT and NC followed by western blot with LAMP2 antibody. (F) A representative western blot of LCA-affinity purified LAMP2. LCA-affinity purification was done in the absence or presence of various concentrations of α-L-fucose (0, 6, 30 and 60 mM). (G) LAMP2 immunoprecipitation from whole-cell lysates of KYSE150 and KYSE450 cells transfected with TSTA3-WT and NC. Anti-LAMP2 immunoprecipitate was treated with or without PNGase F and blotted with biotinylated LCA or LAMP2 antibody. (H) UEA-I affinity chromatography of whole-cell lysate of ESCC cells transfected with TSTA3-WT and NC followed by western blot with ERBB2 antibody. (I) A representative western blot of UEA-I-affinity purified ERBB2. UEA-I-affinity purification was done in the absence or presence of various concentrations of α-L-fucose. (J) ERBB2 immunoprecipitation from whole-cell lysates of KYSE150 and KYSE450 cells transfected with TSTA3-WT and NC. Anti-ERBB2 immunoprecipitate was treated with or without PNGase F and blotted with biotinylated UEA-I or ERBB2 antibody.
    Figure Legend Snippet: Identification of fucosylated glycoproteins in ESCC revealed regulators of invasion and metastasis. (A) Schematic illustration of the experimental approach showing affinity enrichment of core-fucosylated and α-1,2-fucosylated proteins by LCA and UEA-I lectin affinity chromatography respectively. (B) The effect of enriched fucosylated protein by UEA-I lectin with or without α-1,2-fucosidase on KYSE150 and KYSE450 cell invasion. (C) Coomassie brilliant blue (CBB) staining of gels of whole cell lysate proteins and LCA enriched fucosylated proteins in TSTA3-WT and control group. (D) Number of overlapping proteins between lectin enriched proteins identified by in gel mass spectrometry analysis and differentially expressed glycoproteins in N-glycoproteomics data. (E) LCA affinity chromatography of whole-cell lysates of ESCC cells transfected with TSTA3-WT and NC followed by western blot with LAMP2 antibody. (F) A representative western blot of LCA-affinity purified LAMP2. LCA-affinity purification was done in the absence or presence of various concentrations of α-L-fucose (0, 6, 30 and 60 mM). (G) LAMP2 immunoprecipitation from whole-cell lysates of KYSE150 and KYSE450 cells transfected with TSTA3-WT and NC. Anti-LAMP2 immunoprecipitate was treated with or without PNGase F and blotted with biotinylated LCA or LAMP2 antibody. (H) UEA-I affinity chromatography of whole-cell lysate of ESCC cells transfected with TSTA3-WT and NC followed by western blot with ERBB2 antibody. (I) A representative western blot of UEA-I-affinity purified ERBB2. UEA-I-affinity purification was done in the absence or presence of various concentrations of α-L-fucose. (J) ERBB2 immunoprecipitation from whole-cell lysates of KYSE150 and KYSE450 cells transfected with TSTA3-WT and NC. Anti-ERBB2 immunoprecipitate was treated with or without PNGase F and blotted with biotinylated UEA-I or ERBB2 antibody.

    Techniques Used: Affinity Chromatography, Staining, Mass Spectrometry, Transfection, Western Blot, Affinity Purification, Immunoprecipitation

    4) Product Images from "Analysis of serum protein glycosylation by a differential lectin immunosorbant assay (dLISA)"

    Article Title: Analysis of serum protein glycosylation by a differential lectin immunosorbant assay (dLISA)

    Journal: Clinical proteomics

    doi: 10.1186/1559-0275-10-12

    Slopes of the dose response curves established using the dLISA approach correlated with TIMP-1 fucosylation. (A) Dose–response curves of serum samples spiked with differentially fucosylated recombinant TIMP-1. Recombinant TIMP-1 was treated by α1, 2 fucosidase at different length of time (0, 15, 30, and 45 minutes) before they were spiked into with four serum aliquots. These aliquots came from the same pool of sera with endogenous TIMP-1 immuno-depleted (confirmed by the TIMP-1 immunoassay). After the recombinant protein spiked in, these serum samples underwent the dLISA approach. (B) A graph presentation of linear regression slopes of the dose–response curves versus the length of fucosidase treatment (minutes).
    Figure Legend Snippet: Slopes of the dose response curves established using the dLISA approach correlated with TIMP-1 fucosylation. (A) Dose–response curves of serum samples spiked with differentially fucosylated recombinant TIMP-1. Recombinant TIMP-1 was treated by α1, 2 fucosidase at different length of time (0, 15, 30, and 45 minutes) before they were spiked into with four serum aliquots. These aliquots came from the same pool of sera with endogenous TIMP-1 immuno-depleted (confirmed by the TIMP-1 immunoassay). After the recombinant protein spiked in, these serum samples underwent the dLISA approach. (B) A graph presentation of linear regression slopes of the dose–response curves versus the length of fucosidase treatment (minutes).

    Techniques Used: Recombinant

    Related Articles

    Sonication:

    Article Title: Carbohydrate Sequence of the Prostate Cancer-associated Antigen F77 Assigned by a Mucin O-Glycome Designer Array *
    Article Snippet: .. After a brief sonication, 200 units of α1–2 fucosidase (New England Biolabs) were added, and the mixture was incubated at 37 °C for 48 h. The reaction mixture was purified by a C18 cartridge, and the products were eluted with CHCl3 /MeOH/H2 O, 30:70:30. .. Mass Spectrometry MALDI-MS of the oligosaccharides and the derived NGLs was carried out on a TOF Spec-2E (Waters) or an AXIMA Assurance (Shimadzu, Manchester, UK) instrument.

    Incubation:

    Article Title: Carbohydrate Sequence of the Prostate Cancer-associated Antigen F77 Assigned by a Mucin O-Glycome Designer Array *
    Article Snippet: .. After a brief sonication, 200 units of α1–2 fucosidase (New England Biolabs) were added, and the mixture was incubated at 37 °C for 48 h. The reaction mixture was purified by a C18 cartridge, and the products were eluted with CHCl3 /MeOH/H2 O, 30:70:30. .. Mass Spectrometry MALDI-MS of the oligosaccharides and the derived NGLs was carried out on a TOF Spec-2E (Waters) or an AXIMA Assurance (Shimadzu, Manchester, UK) instrument.

    Purification:

    Article Title: Carbohydrate Sequence of the Prostate Cancer-associated Antigen F77 Assigned by a Mucin O-Glycome Designer Array *
    Article Snippet: .. After a brief sonication, 200 units of α1–2 fucosidase (New England Biolabs) were added, and the mixture was incubated at 37 °C for 48 h. The reaction mixture was purified by a C18 cartridge, and the products were eluted with CHCl3 /MeOH/H2 O, 30:70:30. .. Mass Spectrometry MALDI-MS of the oligosaccharides and the derived NGLs was carried out on a TOF Spec-2E (Waters) or an AXIMA Assurance (Shimadzu, Manchester, UK) instrument.

    Isolation:

    Article Title: Site-specific Glycoforms of Haptoglobin in Liver Cirrhosis and Hepatocellular Carcinoma *
    Article Snippet: Two microliters of each exoglycosidase (100 units of neuraminidase and 16 units of galactosidase) were added to the sample based on the manufacturer's recommendation and incubated at 37°C for 20 h. We have verified that each glycosidase reaction reaches completeness by inspection of the spectra (disappearance of the peaks) of known glycoforms. .. For structural characterization of glycopeptides, Hp (2.5 μg) isolated from pooled plasma samples of HCC patients and healthy controls was digested with trypsin as described above, desalted, and treated with exoglycosidases in the following order: α2/3,6,8-neuraminidase (100 units) from C. perfringens overexpressed in E. coli (New England Biolabs); α1/2-fucosidase (20 units) from Xanthomonas manihotis (New England Biolabs); α1/3,4-fucosidase (16 microunits) from almond meal (Prozyme, Hayward, CA); β1,4-galactosidase (16 units) from B. fragilis expressed in E. coli (New England Biolabs); and β1,3-galactosidase (20 units) from X. manihotis expressed in E. coli (New England Biolabs). .. Between each exoglycosidase treatment, glycopeptides were desalted by a microtrap device, eluted with 50% ACN + 0.1% TFA, dried, and an aliquot was resuspended in solvent A (0.1% formic acid in 2% acetonitrile) for MS analysis as described below.

    Recombinant:

    Article Title: Glycan Epitopes on 201B7 Human-Induced Pluripotent Stem Cells Using R-10G and R-17F Marker Antibodies
    Article Snippet: Anti-human iPSC/ESC, R-10G (mouse IgG1), and R-17F (mouse IgG1) antibodies were prepared as described previously [ , ]. .. Peptide N-glycanase (PNGase F; recombinant protein from Escherichia coli ) was obtained from Roche Diagnostics GmbH (Mannheim, Germany), neuraminidase (Arthrobacter ureafaciens ) from Nacalai Tesque (Kyoto, Japan), α1-3/4 fucosidase from TaKaRa Bio, Inc. (Shiga, Japan), and α1-2 fucosidase from New England Biolabs (Ipswich, MA, USA). .. Chondroitinase ABC (Proteus vulgaris ), heparinase mixture (heparinase, heparitinase I, and heparitinase II), keratanase (Pseudomonas sp.), keratanase II (Bacillus sp.), and endo-β-galactosidase (Escherichia freundii) were obtained from Seikagaku Biobusiness (Tokyo, Japan).

    Article Title: Analysis of serum protein glycosylation by a differential lectin immunosorbant assay (dLISA)
    Article Snippet: .. Production of the differential fucosylated recombinant TIMP-1 Four microliter of the recombinant TIMP-1 (10 μg/mL prepared in PBS + 1% BSA buffer) was mixed with 4 μL of α1, 2 fucosidase (Catalog# P0724, New England Biolabs, Ipswich, MA). .. The mixture (8 μL) was then diluted 5 times in the 32 μL of the Reaction buffer (50 mM Sodium Citrate, 100 mM Sodium Chloride, pH 6.0) for incubation of 0, 15, 30, or 45 minutes with shaking at 37°C.

    Blocking Assay:

    Article Title: Bacterial AB5 toxins inhibit the growth of gut bacteria by targeting ganglioside-like glycoconjugates
    Article Snippet: Slides were blocked for 1 h using blocking buffer (PBS containing: 0.2% Triton X-100, 0.05% Tween, 1% bovine serum albumin, 5% donkey serum). .. Where indicated, slides were treated with α1-2-fucosidase (NEB, P0724S) following antigen retrieval in sodium citrate buffer, but prior to blocking with donkey serum buffer. .. Slides were treated for 1 h, with 1 μl (20 units) of α1-2-fucosidase in 10 μl of 1× Glycobuffer (supplied by the manufacturer), at 37 °C, followed by a PBS wash. Each slide was then treated with CTB (1 mM, Sigma, Cat. No. C9903) for 1 h, followed by either α-CTB antibody (1:100) or α-GM1 antibody (1:100, Abcam) for 1 h. Slides were visualized using Alexa Fluor 488-conjugated donkey α-rabbit IgG (1:1000, Invitrogen).

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  • 95
    New England Biolabs fucosidase
    C3d expression and MAC formation on hypoxia-stressed iPS-RPE cells following <t>fucosidase</t> treatment. ( a ) Representative immunofluorescence images showing C3d deposition on the surface of hypoxia-stressed iPS-RPE cells treated or not overnight with fucosidase. C3d (green), nuclei nuclei (blue) and phalloidin (grey) are shown. Scale bars, 50 μm. ( b ) Quantification of C3d expression level on hypoxia-stressed iPS- RPE cells compared to fucosidase-treated hypoxic cells using ImageJ software. ( c ) Representative immunofluorescence images showing MAC staining on the surface of hypoxia-stressed iPS-RPE cells treated or not overnight with fucosidase. MAC (red), nuclei (blue) and phalloidin (grey) are shown. Scale bars, 50 μm.
    Fucosidase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fucosidase/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    fucosidase - by Bioz Stars, 2021-06
    95/100 stars
      Buy from Supplier

    93
    New England Biolabs a1 3 4 fucosidase
    The fucose salvage pathway inhibits invadopodia through α1–2 fucosylation. A, schematic illustration showing the modification of glycans by α-1,2, α-1,3 and α-1,4 branched fucosylations. B, the effects of L-fucose and <t>fucosidase</t> treatment on cell surface α1–2 fucosylation, as determined by UEA-1 staining and flow cytometry. C, quantification of the effects of α-1,2 fucosidase treatment on fucose-mediated inhibition of invadopodium formation in WM793 cells. D, representative invadopodium assay images showing the effect of α-1,2 fucosidase treatment on FUK-mediated inhibition of invadopodium formation in WM793 cells. E and F, quantitation of the effect of α-1,2 fucosidase treatment on the invadopodium number per cell ( E ) and the proportion of invadopodia positive cells ( F ) in control or FUK OE WM793. G and H, quantitation of the effect of <t>α-1,3/4</t> fucosidase treatment on the average invadopodium number per cell ( G ) and proportion of invadopodia positive cells ( H ) in control or fucose-treated WM793. I and J, quantitation of the effect of α-1,3/4 fucosidase treatment on the average invadopodium number per cell ( I ) and proportion of invadopodia positive cells ( J ) in control or FUK-overexpressed WM793. ** and *** indicates p
    A1 3 4 Fucosidase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/a1 3 4 fucosidase/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    a1 3 4 fucosidase - by Bioz Stars, 2021-06
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    Image Search Results


    C3d expression and MAC formation on hypoxia-stressed iPS-RPE cells following fucosidase treatment. ( a ) Representative immunofluorescence images showing C3d deposition on the surface of hypoxia-stressed iPS-RPE cells treated or not overnight with fucosidase. C3d (green), nuclei nuclei (blue) and phalloidin (grey) are shown. Scale bars, 50 μm. ( b ) Quantification of C3d expression level on hypoxia-stressed iPS- RPE cells compared to fucosidase-treated hypoxic cells using ImageJ software. ( c ) Representative immunofluorescence images showing MAC staining on the surface of hypoxia-stressed iPS-RPE cells treated or not overnight with fucosidase. MAC (red), nuclei (blue) and phalloidin (grey) are shown. Scale bars, 50 μm.

    Journal: Scientific Reports

    Article Title: Human stem cell-derived retinal epithelial cells activate complement via collectin 11 in response to stress

    doi: 10.1038/s41598-017-15212-z

    Figure Lengend Snippet: C3d expression and MAC formation on hypoxia-stressed iPS-RPE cells following fucosidase treatment. ( a ) Representative immunofluorescence images showing C3d deposition on the surface of hypoxia-stressed iPS-RPE cells treated or not overnight with fucosidase. C3d (green), nuclei nuclei (blue) and phalloidin (grey) are shown. Scale bars, 50 μm. ( b ) Quantification of C3d expression level on hypoxia-stressed iPS- RPE cells compared to fucosidase-treated hypoxic cells using ImageJ software. ( c ) Representative immunofluorescence images showing MAC staining on the surface of hypoxia-stressed iPS-RPE cells treated or not overnight with fucosidase. MAC (red), nuclei (blue) and phalloidin (grey) are shown. Scale bars, 50 μm.

    Article Snippet: To remove L-fucose from cell surface, iPS-RPE cells were put under hypoxic conditions for 24 hours, fixed in 4% paraformaldehyde for 1 hour at room temperature and treated then with α1-2,3,4,6 fucosidase (New England BioLabs) at 37 °C overnight.

    Techniques: Expressing, Immunofluorescence, Software, Staining

    MALDI-TOF MS analysis of multisulfated complex-type N -glycans. A and B , the highest degree of sulfation of bi/triantennary N -glycans was observed in early-eluting RP HPLC fractions by negative MS in the presence of sodium acetate buffer, which allows detection of [M-H n +Na n-1 ] − ions corresponding to multisulfated structures (structures containing two to four sulfates are indicated in bold ( m/z 2165–3052), and none were digested with a nonspecific galactosidase). Because of in-source loss of sodiated sulfates (Δ 102 ), pseudomolecular ions ( asterisks ) with varying sulfated status ( S1–S4 ) were also observed and fragmented in negative mode as [M-(SO 3 ) n ] − or fragmented in positive mode as [M+H (n-1) -(SO 3 ) n ] + . C and D , analysis in the presence of ammonium sulfate ( C ) results in monosulfated ions, observed in defucosylated form upon fucosidase treatment ( D ). E–J ) were fragmented in negative mode either as in-source pseudomolecular ( F and I ) or parental ions ( G and J ), showing the presence of sulfated Lewis motifs as well as some neutral losses from the core, whereas their bi- and triantennary nature as well as core and antennal fucosylation were confirmed by positive B and Y fragments ( E and H ), such as m/z 446 (Fuc 1 GlcNAc 1 -PA), 512 (Fuc 1 Gal 1 GlcNAc 1 ), and 1484/1646 (Fuc 2 Gal 1 Man 2/3 GlcNAc 3 -PA); the two β2/β4-linked antennae on the lower α3-Man are assumed compared with hybrid N ) of structures predominantly found in later fractions. Losses of Fuc ( F ), Hex ( H ), and HexNAc ( N ) are indicated.

    Journal: The Journal of Biological Chemistry

    Article Title: Sulfated and sialylated N-glycans in the echinoderm Holothuria atra reflect its marine habitat and phylogeny

    doi: 10.1074/jbc.RA119.011701

    Figure Lengend Snippet: MALDI-TOF MS analysis of multisulfated complex-type N -glycans. A and B , the highest degree of sulfation of bi/triantennary N -glycans was observed in early-eluting RP HPLC fractions by negative MS in the presence of sodium acetate buffer, which allows detection of [M-H n +Na n-1 ] − ions corresponding to multisulfated structures (structures containing two to four sulfates are indicated in bold ( m/z 2165–3052), and none were digested with a nonspecific galactosidase). Because of in-source loss of sodiated sulfates (Δ 102 ), pseudomolecular ions ( asterisks ) with varying sulfated status ( S1–S4 ) were also observed and fragmented in negative mode as [M-(SO 3 ) n ] − or fragmented in positive mode as [M+H (n-1) -(SO 3 ) n ] + . C and D , analysis in the presence of ammonium sulfate ( C ) results in monosulfated ions, observed in defucosylated form upon fucosidase treatment ( D ). E–J ) were fragmented in negative mode either as in-source pseudomolecular ( F and I ) or parental ions ( G and J ), showing the presence of sulfated Lewis motifs as well as some neutral losses from the core, whereas their bi- and triantennary nature as well as core and antennal fucosylation were confirmed by positive B and Y fragments ( E and H ), such as m/z 446 (Fuc 1 GlcNAc 1 -PA), 512 (Fuc 1 Gal 1 GlcNAc 1 ), and 1484/1646 (Fuc 2 Gal 1 Man 2/3 GlcNAc 3 -PA); the two β2/β4-linked antennae on the lower α3-Man are assumed compared with hybrid N ) of structures predominantly found in later fractions. Losses of Fuc ( F ), Hex ( H ), and HexNAc ( N ) are indicated.

    Article Snippet: The following glycosidases were employed: recombinant Aspergillus niger β3/4-galactosidase (prepared in-house ( )); Xanthomonas manihotis β3-galactosidase (New England Biolabs); Bacillus fragilis β4-galactosidase (New England Biolabs); bovine kidney α-fucosidase (Sigma-Aldrich); almond α3/4-fucosidase (New England Biolabs); jack bean α-mannosidase (Sigma-Aldrich); purified recombinant Bacteroides xylanisolvens BxGH99 α2-endo-mannosidase, which catalyzes removal of a disaccharide from Glc1 Man9 GlcNAc2 but not from unglucosylated Man9 GlcNAc2 ( ); recombinant Aspergillus saitoi α2-mannosidase (Prozyme); and Streptococcus pneumoniae α3-sialidase S (New England Biolabs).

    Techniques: High Performance Liquid Chromatography

    Identification of fucosylated glycoproteins in ESCC revealed regulators of invasion and metastasis. (A) Schematic illustration of the experimental approach showing affinity enrichment of core-fucosylated and α-1,2-fucosylated proteins by LCA and UEA-I lectin affinity chromatography respectively. (B) The effect of enriched fucosylated protein by UEA-I lectin with or without α-1,2-fucosidase on KYSE150 and KYSE450 cell invasion. (C) Coomassie brilliant blue (CBB) staining of gels of whole cell lysate proteins and LCA enriched fucosylated proteins in TSTA3-WT and control group. (D) Number of overlapping proteins between lectin enriched proteins identified by in gel mass spectrometry analysis and differentially expressed glycoproteins in N-glycoproteomics data. (E) LCA affinity chromatography of whole-cell lysates of ESCC cells transfected with TSTA3-WT and NC followed by western blot with LAMP2 antibody. (F) A representative western blot of LCA-affinity purified LAMP2. LCA-affinity purification was done in the absence or presence of various concentrations of α-L-fucose (0, 6, 30 and 60 mM). (G) LAMP2 immunoprecipitation from whole-cell lysates of KYSE150 and KYSE450 cells transfected with TSTA3-WT and NC. Anti-LAMP2 immunoprecipitate was treated with or without PNGase F and blotted with biotinylated LCA or LAMP2 antibody. (H) UEA-I affinity chromatography of whole-cell lysate of ESCC cells transfected with TSTA3-WT and NC followed by western blot with ERBB2 antibody. (I) A representative western blot of UEA-I-affinity purified ERBB2. UEA-I-affinity purification was done in the absence or presence of various concentrations of α-L-fucose. (J) ERBB2 immunoprecipitation from whole-cell lysates of KYSE150 and KYSE450 cells transfected with TSTA3-WT and NC. Anti-ERBB2 immunoprecipitate was treated with or without PNGase F and blotted with biotinylated UEA-I or ERBB2 antibody.

    Journal: Theranostics

    Article Title: TSTA3 facilitates esophageal squamous cell carcinoma progression through regulating fucosylation of LAMP2 and ERBB2

    doi: 10.7150/thno.48225

    Figure Lengend Snippet: Identification of fucosylated glycoproteins in ESCC revealed regulators of invasion and metastasis. (A) Schematic illustration of the experimental approach showing affinity enrichment of core-fucosylated and α-1,2-fucosylated proteins by LCA and UEA-I lectin affinity chromatography respectively. (B) The effect of enriched fucosylated protein by UEA-I lectin with or without α-1,2-fucosidase on KYSE150 and KYSE450 cell invasion. (C) Coomassie brilliant blue (CBB) staining of gels of whole cell lysate proteins and LCA enriched fucosylated proteins in TSTA3-WT and control group. (D) Number of overlapping proteins between lectin enriched proteins identified by in gel mass spectrometry analysis and differentially expressed glycoproteins in N-glycoproteomics data. (E) LCA affinity chromatography of whole-cell lysates of ESCC cells transfected with TSTA3-WT and NC followed by western blot with LAMP2 antibody. (F) A representative western blot of LCA-affinity purified LAMP2. LCA-affinity purification was done in the absence or presence of various concentrations of α-L-fucose (0, 6, 30 and 60 mM). (G) LAMP2 immunoprecipitation from whole-cell lysates of KYSE150 and KYSE450 cells transfected with TSTA3-WT and NC. Anti-LAMP2 immunoprecipitate was treated with or without PNGase F and blotted with biotinylated LCA or LAMP2 antibody. (H) UEA-I affinity chromatography of whole-cell lysate of ESCC cells transfected with TSTA3-WT and NC followed by western blot with ERBB2 antibody. (I) A representative western blot of UEA-I-affinity purified ERBB2. UEA-I-affinity purification was done in the absence or presence of various concentrations of α-L-fucose. (J) ERBB2 immunoprecipitation from whole-cell lysates of KYSE150 and KYSE450 cells transfected with TSTA3-WT and NC. Anti-ERBB2 immunoprecipitate was treated with or without PNGase F and blotted with biotinylated UEA-I or ERBB2 antibody.

    Article Snippet: Alternatively, samples were treated with fucosidase (NEB) according to the manufacturer's protocol at 37 °C for 4 h.

    Techniques: Affinity Chromatography, Staining, Mass Spectrometry, Transfection, Western Blot, Affinity Purification, Immunoprecipitation

    The fucose salvage pathway inhibits invadopodia through α1–2 fucosylation. A, schematic illustration showing the modification of glycans by α-1,2, α-1,3 and α-1,4 branched fucosylations. B, the effects of L-fucose and fucosidase treatment on cell surface α1–2 fucosylation, as determined by UEA-1 staining and flow cytometry. C, quantification of the effects of α-1,2 fucosidase treatment on fucose-mediated inhibition of invadopodium formation in WM793 cells. D, representative invadopodium assay images showing the effect of α-1,2 fucosidase treatment on FUK-mediated inhibition of invadopodium formation in WM793 cells. E and F, quantitation of the effect of α-1,2 fucosidase treatment on the invadopodium number per cell ( E ) and the proportion of invadopodia positive cells ( F ) in control or FUK OE WM793. G and H, quantitation of the effect of α-1,3/4 fucosidase treatment on the average invadopodium number per cell ( G ) and proportion of invadopodia positive cells ( H ) in control or fucose-treated WM793. I and J, quantitation of the effect of α-1,3/4 fucosidase treatment on the average invadopodium number per cell ( I ) and proportion of invadopodia positive cells ( J ) in control or FUK-overexpressed WM793. ** and *** indicates p

    Journal: PLoS ONE

    Article Title: The fucose salvage pathway inhibits invadopodia formation and extracellular matrix degradation in melanoma cells

    doi: 10.1371/journal.pone.0199128

    Figure Lengend Snippet: The fucose salvage pathway inhibits invadopodia through α1–2 fucosylation. A, schematic illustration showing the modification of glycans by α-1,2, α-1,3 and α-1,4 branched fucosylations. B, the effects of L-fucose and fucosidase treatment on cell surface α1–2 fucosylation, as determined by UEA-1 staining and flow cytometry. C, quantification of the effects of α-1,2 fucosidase treatment on fucose-mediated inhibition of invadopodium formation in WM793 cells. D, representative invadopodium assay images showing the effect of α-1,2 fucosidase treatment on FUK-mediated inhibition of invadopodium formation in WM793 cells. E and F, quantitation of the effect of α-1,2 fucosidase treatment on the invadopodium number per cell ( E ) and the proportion of invadopodia positive cells ( F ) in control or FUK OE WM793. G and H, quantitation of the effect of α-1,3/4 fucosidase treatment on the average invadopodium number per cell ( G ) and proportion of invadopodia positive cells ( H ) in control or fucose-treated WM793. I and J, quantitation of the effect of α-1,3/4 fucosidase treatment on the average invadopodium number per cell ( I ) and proportion of invadopodia positive cells ( J ) in control or FUK-overexpressed WM793. ** and *** indicates p

    Article Snippet: Fucosidase treatment After cell dissociation using enzyme free solution, cells were washed three times in 1xPBS, then incubated in PBS with 0.08U/mL fucosidase (New England Biolabs, α-1,2:P0724S, α-1,3/4:P0769S) for 30 minutes at 37°C.

    Techniques: Modification, Staining, Flow Cytometry, Cytometry, Inhibition, Quantitation Assay