Journal: Cell Discovery

Article Title: Identification of putative novel O-glycosylations in the NK killer receptor Ncr1 essential for its activity

doi: 10.1038/celldisc.2015.36

Figure Lengend Snippet: Ncr1 expressed on primary NK cells carries O-linked glycosylations. ( a ) Western blot (WB) for GAPDH in the whole-cell lysates of purified NK cells derived from WT and Ncr1 gfp/gfp mice before imunoprecipitation. ( b – d ) Ncr1 protein from WT and Ncr1 gfp/gfp mice was immunoprecipitated with the mNcr1.6 mAb, blotted and stained with the mNcr1.5 mAb ( b ), Jacalin (JAC) ( c ) and wheat germ agglutinin (WGA) ( d ) lectins. WB figures were adjusted for better clarity. ( e – g ) Ratio between the GAPDH staining and the mNcr1.5 mAb ( e ) or lectins ( f , g ) staining (quantified by pixel intensity). Values are shown as mean±s.e.m.; * P <0.05. The figures combine three independent experiments.

Article Snippet: For the western blotting with lectins, the various fusion proteins (5 μg) were run on 10% SDS-PAGE gel, transferred to a nitrocellulose membrane (Tamar, Mevaseret Zion, Israel) and blotted with biotin-conjugated lectins (Jacalin, wheat germ agglutinin, Maackia amurensis lectin II and SNA, all from Vector labs, Burlingame, CA, USA) or with biotin-conjugated HA Ig.

Techniques: Western Blot, Purification, Derivative Assay, Immunoprecipitation, Staining

Journal: Cell Discovery

Article Title: Identification of putative novel O-glycosylations in the NK killer receptor Ncr1 essential for its activity

doi: 10.1038/celldisc.2015.36

Figure Lengend Snippet: Thr 222 and Thr 225 are glycosylated. ( a ) Coomassie staining of the various Ncr1 fusion proteins (5 μg) run on 10% SDS-PAGE gel under reducing conditions. ( b – e ) Western blot (WB) performed on the various Ncr1 fusion proteins shown in a . Staining was performed with Jacalin (JAC) ( b ), wheat germ agglutinin (WGA) ( c ), Maackia amurensis lectin II (MAL) ( d ) or Sambucus nigra (SNA) ( e ) lectins. WB and Coomassie figures were adjusted for better clarity. The figures combine at least three independent experiments. ( f – i ) Ratio between the Coomassie staining and the WB staining (quantified by pixel intensity) of the various lectins. Values are shown as mean±s.e.m.; * P <0.05. ( j – n ) HPLC chromatogram of O-linked glycan release from the Ncr1 Ig ( j ), Ncr1 T222A Ig ( k ), Ncr1 T225A Ig ( l ) and Ncr1 T222 225A Ig ( m ) fusion proteins and dextran standards ( n ). The various Ncr1 Ig fusion proteins N-linked glycans were released in solution with PNGase F before O-linked glycan analysis. The data are shown as fluorescence arbitrary units. The chromatogram shown in j is identical to the one shown in Figure 1c albeit presented in a different time scale. The HPLC chromatograms are representatives of two independent runs.

Article Snippet: For the western blotting with lectins, the various fusion proteins (5 μg) were run on 10% SDS-PAGE gel, transferred to a nitrocellulose membrane (Tamar, Mevaseret Zion, Israel) and blotted with biotin-conjugated lectins (Jacalin, wheat germ agglutinin, Maackia amurensis lectin II and SNA, all from Vector labs, Burlingame, CA, USA) or with biotin-conjugated HA Ig.

Techniques: Staining, SDS Page, Western Blot, Fluorescence

Journal: Journal of Virology

Article Title: Syrian Hamster as an Animal Model for the Study of Human Influenza Virus Infection

doi: 10.1128/JVI.01693-17

Figure Lengend Snippet: Detection of SAα2,6Gal and SAα2,3Gal oligosaccharides in the nasal turbinate by using lectins. Sections of a 4-week-old Syrian hamster were reacted with SNA I and MAA II. The vertical lines of the image at the top indicate the anterior surfaces of transverse tissue blocks (A to D). (A) A distal section of the nasal cavity of a 4-week-old hamster showing the predominance of squamous epithelial cells and respiratory epithelial cells. (B to D) The population of olfactory epithelial cells gradually increased from the middle to the deep section of the nasal cavity (B, C); more olfactory epithelial cells than respiratory epithelial cells were present in the deep portion of the nasal cavity (D). SNA I, which is specific for SAα2,6Gal, mainly reacted with respiratory epithelial cells in the distal section of the nasal cavity (A to C); in contrast, MAA II, which is specific for SAα2,3Gal, mainly reacted with olfactory epithelial cells in the proximal portion of the nasal turbinates of hamsters (C, D). HE, hematoxylin and eosin staining.

Article Snippet: They were then incubated at 4°C overnight with biotin-conjugated SNA I (EY Laboratories) for SAα2,6Gal detection and biotinylated conjugated MAA II (Vector Laboratories) for SAα2,3Gal detection.

Techniques: Staining

Journal: Journal of Virology

Article Title: Syrian Hamster as an Animal Model for the Study of Human Influenza Virus Infection

doi: 10.1128/JVI.01693-17

Figure Lengend Snippet: Detection of SAα2,6Gal and SAα2,3Gal oligosaccharides in the pharynx (A), trachea (B), bronchus (C), and bronchiole/alveolar region (D) of a 4-week-old Syrian hamster. In the pharynx, trachea, and bronchus, both SNA I and MAA II strongly reacted with the epithelial cells (A, B, C). In contrast, MAA II strongly reacted with the epithelial cells in the lungs (D). HE, hematoxylin and eosin staining.

Article Snippet: They were then incubated at 4°C overnight with biotin-conjugated SNA I (EY Laboratories) for SAα2,6Gal detection and biotinylated conjugated MAA II (Vector Laboratories) for SAα2,3Gal detection.

Techniques: Staining

Journal: Cancer Immunology, Immunotherapy

Article Title: Expression profiling of immune inhibitory Siglecs and their ligands in patients with glioma

doi: 10.1007/s00262-019-02332-w

Figure Lengend Snippet: Expression of sialic acids on blood MDSCs from healthy donors and glioma patients. a , b Binding of sialic acid-binding lectins MALII (α2,3-linked sialic acid) and SNA-I (α2,6-linked sialic acid) and galactose-binding lectin PNA to MDSCs isolated from blood of healthy donors. a Representative histograms show lectin binding to M-MDSCs (upper panel) and PMN-MDSCs (lower panel) as determined by flow cytometry. The gray histograms represent the unstained control b ) Bar diagram shows MFI ± SEM of lectin binding to both MDSC subsets ( n = 3). c , d Binding of recombinant Siglec Fc chimera to MDSCs from healthy donors ( n = 3). Bar diagrams shows MFI ± SEM of Siglec Fc binding to M-MDSCs ( c ) and PMN-MDSCs ( d ). e , f Siglec ligand expression on PMN-MDSCs obtained from the blood of glioma patients ( n = 5). Bar diagram shows Siglec Fc binding as MFI ± SEM ( e ) and histograms show representative Siglec Fc binding ( f ). The gray histograms represent the isotype control

Article Snippet: For lectin staining, cells were stained with biotinylated lectins Maackia amurensis lectin II (MALII), Sambucus nigra agglutinin (SNA-I) and peanut agglutinin (PNA) in carbo-free blocking solution containing 1 mM CaCl 2+ and 1 mM MgCl 2+ (Vector Laboratories, Burlington, NO) for 40 min. After washing, the cells were incubated with PE-conjugated Streptavidin (BD, Franklin Lakes, NJ).

Techniques: Expressing, Binding Assay, Isolation, Flow Cytometry, Recombinant

Journal: bioRxiv

Article Title: High mannose N-glycans on red blood cells as phagocytic ligands, mediating both sickle cell anaemia and resistance to malaria

doi: 10.1101/2020.11.26.399402

Figure Lengend Snippet: a) GNA lectin western blot from healthy (HbAA) and sickle (HbSS) ghosts. b) Above are shown further GNA lectin western blots from HbAA and HBSS ghosts. The histogram below the blot shows the flow cytometrically measured surface GNA lectin staining values of the RBCs used to make the ghosts, with each bar corresponding to the cells used to make the western lane above. The r value to the right of the 100kDa size label is Spearman’s rank correlation coefficient between GNA lectin staining values and band intensities, both classified ordinally as high, medium or low (n=27 measurements from 22 individuals). None of the other bands yielded significant correlation coefficients. c) GNA lectin blot from HbSS ghosts: untreated (U), treated by PNGase (P) or Endo-H (E). d) High exposure β-spectrin blot showing PNGase and partial/full (0.1X/1X) Endo-H digestion of two HbSS ghosts. e) Lectin precipitation of healthy ghosts with GNA or MAL-II lectins. No lectin control is also shown. Immunoblot with β-spectrin specific antibody. f) Super resolution microscopy image of spectrin membrane skeleton (blue) from healthy (AA) and sickle cells (SS). Yellow clusters of GNA staining are overlaid. 3D image is sliced to reveal single sheet of membrane skeleton network. g) GNA lectin blot of spectrin released from HbSS ghosts after digestion with trypsin for one hour. Untreated (UT), Tx indicates the dilution factor of trypsin relative to spectrin material. h) Peptide coverage and intensity map of α-spectrin from proteomic analysis of F40 following chymotrypsin treatment. i) GNA lectin blots showing Endo-H treatment of the 10kDa concentrate from (e) under native or denaturing conditions (urea/SDS/2-mercaptoethanol) for 24 hours. j) 3D SIM super-resolution microscopy of surface GNA lectin binding and internal β-spectrin in HbSS. HbSS RBC are first stained with GNA lectin (yellow), then permeabilized, and stained with anti-spectrin antibody (blue).

Article Snippet: They include: Galanthus nivalis Agglutinin (GNA, B-1245, 4μg/ml), Narcissus pseudonarcissus Lectin (NPL, B-1375, 4μg/ml), Griffonia simplicifolia Lectin II (G.Simp, B-1215, 4μg/ml), Solanum tuberosum Lectin (STL, B-1165, 20 ng/ml), Aleuria aurantia Lectin (AAL, B-1395, 33ng/ml), Maackia amurensis Lectin II (MAL II, B-1265, 67ng/ml), Sophora japonica Lectin (Vector Laboratories, no longer available, 1μg/ml).

Techniques: Western Blot, Staining, Microscopy, Binding Assay

Journal: Scientific Reports

Article Title: Variability of serum IgG sialylation and galactosylation degree in women with advanced endometriosis

doi: 10.1038/s41598-021-85200-x

Figure Lengend Snippet: Relative reactivities of serum native IgG (s-IgG)—( a ), and isolated serum IgG (i-IgG)—( b ) glycans with specific lectins. GSL-II: Griffonia simplicifolia lectin II, recognizing terminal GlcNAc; RCA-I: Ricinus communis agglutinin I, recognizing terminal Gal; MAA: Maackia amurensis agglutinin, recognizing sialic acid α2,3-linked; SNA: Sambucus nigra agglutinin, recognizing sialic acid α2,6-linked. E—endometriosis, NE—no endometriosis and Control—group of healthy women. The relative reactivities with lectins were expressed in absorbance units (AU). ( c )—the values of agalactosylation factor GSL-II/RCA-I for IgG glycans. ( d )—the values of MAA/SNA ratio for IgG glycans. MAA: Maackia amurensis agglutinin, recognizing sialic acid α2,3-linked; SNA: Sambucus nigra agglutinin, recognizing sialic acid α2,6-linked. Significant differences versus groups: 1 E, 2 NE. Median is indicated as square. A two-tailed p -value of less than 0.05 was considered as significant.

Article Snippet: The method was based on the reactivity of IgG glycan moieties with specific biotinylated lectins: Maackia amurensis agglutinin (MAA, recognizing sialic acid α2,3-linked) and Sambucus nigra agglutinin (SNA, which recognize sialic acid α2,6-linked), Ricinus communis agglutinin I (RCA-I) which detect the terminal galactose; Griffonia simplicifolia lectin II (GSL-II) detecting the terminal GlcNAc (Vector Laboratories Inc., Burlingame, CA, USA).

Techniques: Isolation, Two Tailed Test