Journal: bioRxiv

Article Title: Activity-dependent Golgi satellite formation in dendrites reshapes the neuronal surface glycoproteome

doi: 10.1101/2021.04.06.438745

Figure Lengend Snippet: Nicotine exposure induces the modification of α4β2R N-linked glycans to complex forms. A . Endo H and PNGase F cleavage of surface α4 subunits from untreated or nicotine-treated α4β2R cells. Cells were untreated or nicotine-treated for 17 hours. Proteins on the cell surface were then biotinylated, solubilized, precipitated with stepavidin-agarose and glycosidase-treated with Endo H or PNGase F enzymes on the agarose. Afterwards, eluted proteins were analyzed on SDS-PAGE and immunoblotted using anti-α4 antibody. B . Swainsonine treatment blocks nicotine-induced glycan modification of surface α4 subunits. α4β2R cells were treated with the α-mannosidase inhibitor, swainsonine, for 2 hours and then swainsonine and nicotine for 17 hours. Afterwards, samples were prepared as in A. C . Neuraminidase (NMdase) cleavage of surface α4 subunits from untreated (-nicotine) or nicotine-treated (+nicotine) α4β2R cells. α4β2R cells were prepared as in A except with NMdase, which cleaves sialic acid, replacing the other glycosidase enzymes. D . Sambucus Nigra (SNA) lectin recognizes α4 subunits after nicotine treatment. Samples were prepared as in A, and after solubilization, proteins were precipitated with the agarose-conjugated sialic acid-recognizing, lectin SNA and then analyzed by immunoblotting with anti-α4 antibody (left) and compared to samples precipitated with with stepavidin-agarose as in A-C (right). E . Ammonium chloride treatment blocks nicotine-induced glycan modification of surface α4 subunits. α4β2 cells were treated with or without ammonium chloride (NH 4 Cl; 10 mM) to inhibit activity of sialyltransferases that require an acidic environment to function. Samples were processed as in B, with NH 4 Cl replacing swainsonine. F . Time course of surface α4 subunit glycan modification. α4β2R cells were nicotine treated (+Nic) or untreated (-Nic) for displayed times, after which the cells were surface biotinylated and surface α4 subunits processed as in the previous panels. G . Quantification of the time course of surface α4 subunit glycan modification. Densitometry of α4 subunit bands from 3 separate experiments, as in , was preformed and the ratio of complex-trimmed (upper) band to the high-mannose (lower) bands are plotted as the mean ± the SEM.

Article Snippet: For the SNA lectin pull down experiments, lysates from α4β2R cells were incubated with agarose-linked Sambucus Nigra lectin, SNA (100 μl; Vector Laboratories) with rotation at 4°C overnight.

Techniques: Modification, SDS Page, Western Blot, Activity Assay

Journal: bioRxiv

Article Title: Sialidases derived from Gardnerella vaginalis remodel the sperm glycocalyx and impair sperm function

doi: 10.1101/2025.02.01.636076

Figure Lengend Snippet: A) Schematic describing lectin flow cytometry and representative glycan epitopes. B, C, D) Sperm were exposed to sialidases (AUS 20 units or GvNanH2 160 units) for 1 hour, fixed in formalin, and stained with MAL-II-biotin/Neutravidin-Alexa Fluor 488, SNA-Cy5, or PSA-FITC respectively. Each point represents the median fluorescence intensity from one donor. Statistics represent a mixed model comparison with post-hoc Holm-Šidák tests. E) Schematic representing change in zeta potential after removal of negatively charged sialic acid on sperm surface. F) Sperm were treated with sialidase at various doses for 1 hour, and zeta potential was measured via dynamic light scattering. Each measurement represents the average of seven technical replicates, and statistics represent a mixed model comparison with post hoc Holm-Šidák tests.

Article Snippet: Biotinylated Maackia Amurensis Lectin II (MAL II, Cat #:B-1265-1) and Cy5-conjugated Sambucus Nigra Lectin (SNA, Cat #: CL-1305-1) were purchased from Vector Labs. Human Contraception Antibody (HCA), was a gift from ZabBio.

Techniques: Flow Cytometry, Staining, Fluorescence, Comparison, Zeta Potential Analyzer

Journal: Cancers

Article Title: Decoding Single Cell Morphology in Osteotropic Breast Cancer Cells for Dissecting Their Migratory, Molecular and Biophysical Heterogeneity

doi: 10.3390/cancers14030603

Figure Lengend Snippet: Differential binding of lectins to breast cancer cell lines. ( A , B ) The parental cells MB-231 and its bone-seeking derivatives, MET and BONE, were cell-surface labeled with one of a panel of distinct FITC-conjugated lectins prior to cytochemistry (( A ), left panels) and flow cytometry (( A ), right panels, ( B )) analyses. See also . Percentages of positive cells are indicated in the histograms ( A ) and the median fluorescence intensity is presented for each lectin ( B ). Data from representative experiments acquired under uniform instruments setting for all cell lines are displayed. The lectins used were Concanavalin A (ConA), Datura Stramonium Lectin (DSL), Dolichos Biflorus Agglutinin (DBA), Erythrina Cristagalli Lectin (ECL), Griffonia Simplicifolia Lectin I (GSL-I), Griffonia Simplicifolia Lectin II (GSL-II), Lens Culinaris Agglutinin (LCA), Lycopersicon Esculentum Lectin (LEL), Peanut Agglutinin (PNA), Phaseolus Vulgaris Lectin E (PHA-E), Phaseolus Vulgaris Lectin L (PHA-L), Pisum Sativum Agglutinin (PSA), Ricinus Communis Agglutinin I (RCA), Sambucus Nigra Lectin (SNA), Solanum Tuberosum Lectin (STL), Soybean Agglutinin (SBA), Ulex Europaeus Agglutinin I (UEA-I), Vicia Villosa Lectin (VVL) and Wheat Germ Agglutinin (WGA, succinylated (succ)-WGA). Scale bar, 25 μm.

Article Snippet: After inactivation of trypsin, 2 washing steps with PBS and centrifugation (5 min at 300× g ), cells were resuspended in PBS or Ca/Mg-PBS (for lectin labeling), containing 1% BSA and 100 μL-cell suspension aliquots were incubated with unconjugated or fluorochrome-conjugated primary antibodies ( ) or FITC- or biotin-conjugated lectins (see above, Lectin kit I, Biotinylated (BK-1000) and SNA, Biotinylated (B-1305-2), both from Vector Laboratories) for 30 min at 4 °C.

Techniques: Binding Assay, Labeling, Flow Cytometry, Fluorescence

Journal: Frontiers in Microbiology

Article Title: Capsular Sialyltransferase Specificity Mediates Different Phenotypes in Streptococcus suis and Group B Streptococcus

doi: 10.3389/fmicb.2018.00545

Figure Lengend Snippet: Capsular polysaccharide (CPS) expression levels and sialic acid linkage in S. suis serotype 2 and 14 mutants carrying exogenous α-2,3-sialyltransferase. (A) Hydrophobicity (%) of the wild-type S. suis serotype 2 (SS2) and 14 (SS14) strains, the SS2sia2,3 (Δ cps2N / cpsK ) and SS14sia2,3 (Δ cps14N / cpsK ) mutants carrying the GBS α-2,3-sialyltransferase ( cpsK ). The non-encapsulated mutants SS2Δ cps and SS14Δ cps were used as control strains. (B,C) Whole-bacterial cell enzyme-linked lectin assay (ELLA) was performed to detect α-2,3 or α-2,6 capsular sialic acid linkage in SS2sia2,3 and SS14sia2,3 mutant strains. Whole bacteria were incubated with Sambucus nigra agglutinin (SNA-I) specific for Neu5Ac α-2,6 linkages, or Maackia amurensis leukoagglutinin (MAL-I) specific for Neu5Ac α-2,3 linkages. The non-encapsulated mutants SS2Δ cps and SS14Δ cps were used as negative controls. SS2 was used as positive control for SNA-I and wild-type GBS type III as positive control for MAL-I. Data in (A–C) are expressed as mean ± SEM of at least three independent experiments. Student's t -test analyses reported significant differences between “ a” and “ b” ( P < 0.05).

Article Snippet: In order to investigate the specific linkage of sialic acid in the sialyltransferase substitution mutants, a whole-bacterial cell ELLA was carried out with the biotinylated Sambucus nigra agglutinin (SNA-I, Vector Labs Canada, Burlington, ON, Canada) and the biotinylated Maackia amurensis leukoagglutinin (MAL-I, Vector Labs) which specifically recognize sialic acid as Neu5Acα-2,6-Gal p /Gal p NAc or as Neu5Acα-2,3-Galβ-1,4-GlcNAc, respectively (Shibuya et al., ; Geisler and Jarvis, ).

Techniques: Expressing, Mutagenesis, Incubation, Positive Control

Journal: Frontiers in Microbiology

Article Title: Capsular Sialyltransferase Specificity Mediates Different Phenotypes in Streptococcus suis and Group B Streptococcus

doi: 10.3389/fmicb.2018.00545

Figure Lengend Snippet: Capsular polysaccharide (CPS) expression levels and recognition of specific CPS sialic acid linkage in GBS type V isogenic mutants. (A) Hydrophobicity (%) of the wild-type GBS serotype V strain (GBS V), and the sialic acid synthesis GBSVΔsynth (Δ neu5B ) and sialyltransferase GBSVΔsiaT (Δ cps5K ) deficient mutants. The non-encapsulated strain (GBSVΔ cps ) was used as control. (B) Whole-bacterial cell enzyme-linked lectin assay (ELLA) was performed to detect α-2,3 or α-2,6 capsular sialic acid linkage in these mutant strains. Whole bacteria were incubated with Sambucus nigra agglutinin (SNA-I) specific for Neu5Ac α-2,6 linkages, or Maackia amurensis leukoagglutinin (MAL-I) specific for Neu5Ac α-2,3 linkages. The non-encapsulated mutant GBSVΔ cps was used as negative control. S. suis serotype 2 (SS2) was used as positive control for SNA-I and wild-type GBS type V as positive control for MAL-I. Data in (A,B) are expressed as mean ± SEM of at least three independent experiments. Student's t -test analyses reported significant differences between “ a” and “ b” ( P < 0.05).

Article Snippet: In order to investigate the specific linkage of sialic acid in the sialyltransferase substitution mutants, a whole-bacterial cell ELLA was carried out with the biotinylated Sambucus nigra agglutinin (SNA-I, Vector Labs Canada, Burlington, ON, Canada) and the biotinylated Maackia amurensis leukoagglutinin (MAL-I, Vector Labs) which specifically recognize sialic acid as Neu5Acα-2,6-Gal p /Gal p NAc or as Neu5Acα-2,3-Galβ-1,4-GlcNAc, respectively (Shibuya et al., ; Geisler and Jarvis, ).

Techniques: Expressing, Mutagenesis, Incubation, Negative Control, Positive Control

Journal: Frontiers in Microbiology

Article Title: Capsular Sialyltransferase Specificity Mediates Different Phenotypes in Streptococcus suis and Group B Streptococcus

doi: 10.3389/fmicb.2018.00545

Figure Lengend Snippet: Capsular polysaccharide (CPS) expression levels and sialic acid linkage in GBS type III and V mutants carrying exogenous α-2,6-sialyltransferase. (A) Hydrophobicity (%) of GBS type III and V wild-type strains and the mutants GBSIIIsia2,6 (Δ cps3K / cps2N ) and GBSVsia2,6 (Δ cps5K / cps2N ) carrying the S. suis α-2,6-sialyltransferase. The non-encapsulated mutant strains (GBSIIIΔ cps and GBSVΔ cps ) were used as controls. (B,C) Whole-bacterial cell enzyme-linked lectin assay (ELLA) was performed to detect α-2,3 or α-2,6 capsular sialic acid linkage in these mutant strains. Whole bacteria were incubated with Sambucus nigra agglutinin (SNA-I) specific for Neu5Ac α-2,6 linkages, or Maackia amurensis leukoagglutinin (MAL-I) specific for Neu5Ac α-2,3 linkages. The non-encapsulated mutants were used as negative controls. Data in (A–C) are expressed as mean ± SEM of at least three independent experiments. Student's t -test analyses reported significant differences between “ a” and “ b” , between “ a” and “ c” , and between “ b” and “ c” ( P < 0.05).

Article Snippet: In order to investigate the specific linkage of sialic acid in the sialyltransferase substitution mutants, a whole-bacterial cell ELLA was carried out with the biotinylated Sambucus nigra agglutinin (SNA-I, Vector Labs Canada, Burlington, ON, Canada) and the biotinylated Maackia amurensis leukoagglutinin (MAL-I, Vector Labs) which specifically recognize sialic acid as Neu5Acα-2,6-Gal p /Gal p NAc or as Neu5Acα-2,3-Galβ-1,4-GlcNAc, respectively (Shibuya et al., ; Geisler and Jarvis, ).

Techniques: Expressing, Mutagenesis, Incubation