Journal: Cellular and Molecular Life Sciences

Article Title: Defective IGF-1 prohormone N-glycosylation and reduced IGF-1 receptor signaling activation in congenital disorders of glycosylation

doi: 10.1007/s00018-022-04180-x

Figure Lengend Snippet: Lectin-binding analysis. Concanavalin A (ConA) and Phaseolus vulgaris leucoagglutinin (PHA-L) binding to CTR (mean of three technical replicates for each of five biological replicates) and CDG (mean of three technical replicates for each of twelve biological replicates) fibroblasts. Con A and PHA-L recognize high mannose and complex type N-glycans, respectively. *significantly different from CTR fibroblasts, * p < 0.0001

Article Snippet: For lectin blotting, membranes were probed with biotinylated Concanavalin A (ConA, 1:1000; Cat #B-1005-5) and Phaseolus vulgaris leucoagglutinin (PHA-L, 1:200; Cat #B-1115-2) lectins (Vector laboratories, D.B.A.

Techniques: Binding Assay

Journal: Journal of the American Chemical Society

Article Title: The Metabolic Chemical Reporter 6‑Azido-6-deoxy-glucose Further Reveals the Substrate Promiscuity of O ‑GlcNAc Transferase and Catalyzes the Discovery of Intracellular Protein Modification by O ‑Glucose

doi: 10.1021/jacs.7b13488

Figure Lengend Snippet: A significant fraction of 6AzGlc-dependent labeling is O-linked. (A) Known O-GlcNAcylated proteins are labeled by 6AzGlc. H1299 cells were treated with either Ac46AzGlc (200 μM) or DMSO for 16 h, followed by CuAAC with a cleavable alkyne-biotin tag. After enrichment on streptavidin beads, the labeled proteins were eluted and visualized by Western blotting. The nonglycosylated protein β-actin is a negative control. (B) A notable fraction of 6AzGlc-dependent signal is sensitive to β-elimination. NIH3T3 cells were treated with either Ac46AzGlc (200 μM) or DMSO vehicle for 16 h, followed by CuAAC with alkyne-biotin, separation by SDS-PAGE and transfer to a PVDF membrane. The indicated membranes were then treated for 24 h with either H2O or 55 mM NaOH before analysis by streptavidin or Western blotting. (C) 6AzGlc is not incorporated into N-linked glycans. NIH3T3 cells were treated with either Ac46AzGlc (200 μM) or DMSO vehicle for 16 h. The corresponding cell lysates were then incubated with either PNGase-F or H2O vehicle as indicated before CuAAC with alkyne TAMRA and analysis by in-gel fluorescence. A fraction of the treated lysate was separated before CuAAC and analyzed by Lectin blotting with Concanavalin A (ConA).

Article Snippet: The blots washed three times in TBST for 5 min and incubated with biotin-conjugated Concanavalin A (Vector Lab), diluted 1:1000 in TBST, for 1 h. The blot was then washed 3× with TBST for 10 min. Then the blot was then incubated with Strep-HRP at 1:1000 in blocking buffer for 1 h. After being washed 3× with TBST for 10 min, the blot was developed using ECL reagents.

Techniques: Labeling, Western Blot, Negative Control, SDS Page, Incubation, Fluorescence

Journal:

Article Title: Identification of an Intestinal Neutral Glycosphingolipid as a Phenotype-Specific Receptor for the K88ad Fimbrial Adhesin of Escherichia coli †

doi:

Figure Lengend Snippet: Binding of K88ad adhesin to neutral glycosphingolipids from adhesive and nonadhesive phenotypes of pigs. Neutral glycosphingolipids (100 μg) from five phenotypes of pigs (A [lane 2], E [lane 3], B [lane 4], C [lane 5], and D [lane 6]) were separated on HPTLC plates as described in Materials and Methods. Chromatograms were either stained with orcinol-sulfuric acid reagent (panel I) or incubated with biotinylated K88ad adhesin (panel II) as described in Materials and Methods. Lane 1 contains glycolipid standards Lc2Cer (2), Gb4Cer (4), and Gb5Cer (5). The arrowheads indicate the positions of IGLad.

Article Snippet: Biotinylated vegetable lectins (concanavalin A lectin [ConA], soybean agglutinin [SBA], peanut agglutinin [PNA], Sambucus nigra agglutinin [SNA], Bandeira simplicifolia lectin [BSL II], Datura stramonium lectin [DSL], Jacalin, wheat germ agglutinin [WGA], Ulex europaeus agglutinin [UEA], Ricinus communis agglutinin [RCA 120 ], Maackia amurensis lectin I and II [MAL I and MAL II], Erythrina cristagalli lectin [ECL], Vicia villosa agglutinin [VVA]) were obtained from Vector Laboratories, Inc. (Burlingame, Calif.).

Techniques: Binding Assay, High Performance Thin Layer Chromatography, Staining, Incubation

Journal:

Article Title: Identification of an Intestinal Neutral Glycosphingolipid as a Phenotype-Specific Receptor for the K88ad Fimbrial Adhesin of Escherichia coli †

doi:

Figure Lengend Snippet: Determination of the binding of selected biotinylated lectins

Article Snippet: Biotinylated vegetable lectins (concanavalin A lectin [ConA], soybean agglutinin [SBA], peanut agglutinin [PNA], Sambucus nigra agglutinin [SNA], Bandeira simplicifolia lectin [BSL II], Datura stramonium lectin [DSL], Jacalin, wheat germ agglutinin [WGA], Ulex europaeus agglutinin [UEA], Ricinus communis agglutinin [RCA 120 ], Maackia amurensis lectin I and II [MAL I and MAL II], Erythrina cristagalli lectin [ECL], Vicia villosa agglutinin [VVA]) were obtained from Vector Laboratories, Inc. (Burlingame, Calif.).

Techniques: Binding Assay

Journal:

Article Title: Identification of an Intestinal Neutral Glycosphingolipid as a Phenotype-Specific Receptor for the K88ad Fimbrial Adhesin of Escherichia coli †

doi:

Figure Lengend Snippet: Binding of K88ad adhesin to intestinal neutral glycosphingolipids after β-galactosidase treatment. Neutral glycosphingolipids from a phenotype A animal were treated with β-galactosidase as described in Materials and Methods. Both treated (lane 3) and untreated (lane 2) glycosphingolipids (100 μg) were separated on HPTLC plates as described in Materials and Methods. Chromatograms were stained with orcinol-sulfuric acid reagent (A), or incubated with biotinylated K88ad adhesin (B) or RCA120 (C) as described in Materials and Methods. Lane 1 contains glycolipid standards Lc2Cer (2), Gb4Cer (4) and Gb5Cer (5). The arrowheads indicate the positions of IGLad.

Article Snippet: Biotinylated vegetable lectins (concanavalin A lectin [ConA], soybean agglutinin [SBA], peanut agglutinin [PNA], Sambucus nigra agglutinin [SNA], Bandeira simplicifolia lectin [BSL II], Datura stramonium lectin [DSL], Jacalin, wheat germ agglutinin [WGA], Ulex europaeus agglutinin [UEA], Ricinus communis agglutinin [RCA 120 ], Maackia amurensis lectin I and II [MAL I and MAL II], Erythrina cristagalli lectin [ECL], Vicia villosa agglutinin [VVA]) were obtained from Vector Laboratories, Inc. (Burlingame, Calif.).

Techniques: Binding Assay, High Performance Thin Layer Chromatography, Staining, Incubation

Journal:

Article Title: Identification of an Intestinal Neutral Glycosphingolipid as a Phenotype-Specific Receptor for the K88ad Fimbrial Adhesin of Escherichia coli †

doi:

Figure Lengend Snippet: Binding of K88ad adhesin to intestinal neutral glycosphingolipids after α-fucosidase treatment. Neutral glycosphingolipids from a phenotype A animal were treated with α-fucosidase as described in Materials and Methods. Both treated (lane 3) and untreated (lane 2) glycosphingolipids (100 μg) were separated on HPTLC plates as described in Materials and Methods. Chromatograms were stained with orcinol-sulfuric acid reagent (A) or incubated with biotinylated K88ad adhesin (B) or UEA (C) as described in Materials and Methods. Lane 1 contains glycolipid standards Lc2Cer (2), Gb4Cer (4) and Gb5Cer (5). The arrowheads indicate the positions of IGLad.

Article Snippet: Biotinylated vegetable lectins (concanavalin A lectin [ConA], soybean agglutinin [SBA], peanut agglutinin [PNA], Sambucus nigra agglutinin [SNA], Bandeira simplicifolia lectin [BSL II], Datura stramonium lectin [DSL], Jacalin, wheat germ agglutinin [WGA], Ulex europaeus agglutinin [UEA], Ricinus communis agglutinin [RCA 120 ], Maackia amurensis lectin I and II [MAL I and MAL II], Erythrina cristagalli lectin [ECL], Vicia villosa agglutinin [VVA]) were obtained from Vector Laboratories, Inc. (Burlingame, Calif.).

Techniques: Binding Assay, High Performance Thin Layer Chromatography, Staining, Incubation

Journal:

Article Title: Identification of an Intestinal Neutral Glycosphingolipid as a Phenotype-Specific Receptor for the K88ad Fimbrial Adhesin of Escherichia coli †

doi:

Figure Lengend Snippet: Binding of K88ad adhesin to purified neutral glycosphingolipids. Two micrograms of Lc3Cer (lane 1), Lc3Cer plus Lc4Cer (lane 2), nLc4Cer (lane 3), III4FucLc4Cer or Lea (lane 4), III3FucnLc4Cer or Lex (lane 5), V3FucnLc6Cer (lane 6), and VI2FucnLc6Cer (lane 7) were separated on HPTLC. These glycosphingolipids were either stained with orcinol-sulfuric acid reagent (A) or incubated with biotinylated K88ad adhesin (B) as described in Materials and Methods.

Article Snippet: Biotinylated vegetable lectins (concanavalin A lectin [ConA], soybean agglutinin [SBA], peanut agglutinin [PNA], Sambucus nigra agglutinin [SNA], Bandeira simplicifolia lectin [BSL II], Datura stramonium lectin [DSL], Jacalin, wheat germ agglutinin [WGA], Ulex europaeus agglutinin [UEA], Ricinus communis agglutinin [RCA 120 ], Maackia amurensis lectin I and II [MAL I and MAL II], Erythrina cristagalli lectin [ECL], Vicia villosa agglutinin [VVA]) were obtained from Vector Laboratories, Inc. (Burlingame, Calif.).

Techniques: Binding Assay, Purification, High Performance Thin Layer Chromatography, Staining, Incubation

Journal:

Article Title: Identification of an Intestinal Neutral Glycosphingolipid as a Phenotype-Specific Receptor for the K88ad Fimbrial Adhesin of Escherichia coli †

doi:

Figure Lengend Snippet: Comigration of IGLad and nLc4Cer on HPTLC plates. Neutral glycosphingolipids from a phenotype A animal (100 μg, lane 2), and nLc4Cer (2 μg, lane 3) were separated on HPTLC. These glycosphingolipids were either stained with orcinol-sulfuric acid reagent (A) or incubated with biotinylated K88ad adhesin (B) as described in Materials and Methods. Lane 1 contains glycolipid standards (Lc2Cer [2], Gb4Cer [4], and Gb5Cer [5]). The arrowheads indicate the position of IGLad.

Article Snippet: Biotinylated vegetable lectins (concanavalin A lectin [ConA], soybean agglutinin [SBA], peanut agglutinin [PNA], Sambucus nigra agglutinin [SNA], Bandeira simplicifolia lectin [BSL II], Datura stramonium lectin [DSL], Jacalin, wheat germ agglutinin [WGA], Ulex europaeus agglutinin [UEA], Ricinus communis agglutinin [RCA 120 ], Maackia amurensis lectin I and II [MAL I and MAL II], Erythrina cristagalli lectin [ECL], Vicia villosa agglutinin [VVA]) were obtained from Vector Laboratories, Inc. (Burlingame, Calif.).

Techniques: High Performance Thin Layer Chromatography, Staining, Incubation

Journal: Cell

Article Title: Small RNAs are modified with N-glycans and displayed on the surface of living cells

doi: 10.1016/j.cell.2021.04.023

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: All lectins were bought biotinylated from Vector labs: biotin-wheat germ agglutinin (WGA), biotin-concanavalin A (ConA), and biotin-Maackia Amurensis Lectin II (MAAII).

Techniques: Recombinant, Staining, Blocking Assay, Plasmid Preparation, High Performance Liquid Chromatography, Protein Extraction, Expressing, Software