fucosidase  (New England Biolabs)

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

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

Journal: The Journal of Biological Chemistry

Article Title: Carbohydrate Sequence of the Prostate Cancer-associated Antigen F77 Assigned by a Mucin O-Glycome Designer Array *

doi: 10.1074/jbc.M114.558932

Figure Lengend Snippet: MALDI-MS analysis of NGL 4M-f1 before ( A ) and after ( B ) treatment with α1–2 fucosidase. The change in molecular ion [MH] + from m / z 2035 ( A ) to 1889 ( B ) indicated the capping sequence Fucα1–2Gal in 4M-f1. Abbreviations are as in legend to Fig. 4 .

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.

Techniques: Mass Spectrometry, Sequencing

Journal: Clinical proteomics

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

doi: 10.1186/1559-0275-10-12

Figure Lengend 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).

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).

Techniques: Recombinant