alexa fluor  (Vector Laboratories)

Journal: International Journal of Medical Sciences

Article Title: POFUT2 Mediated Fucosylation of JUP Enhances VEGFA Expression to Promote Angiogenesis in Colorectal Cancer

doi: 10.7150/ijms.113515

Figure Lengend Snippet: POFUT2 mediated fucosylation of JUP to enhance VEGFA expression. (A) Detection of JUP fucosylation by Western Blot after POFUT2 overexpression in HCT8 cells, using an exogenous Flag antibody to pull down POFUT2, followed by lectin AAL blotting. (B) Detection of JUP expression level after POFUT2 overexpression in HCT8 cells with a 24-hour treatment of SGN-2FF (10μm). (C) Scatterplot illustrating the expression correlation between JUP and VEGFA within the TCGA CRC cohort. (D) qRT-PCR detection of VEGFA expression following JUP knockdown in HCT8 cells. (E) Western blot analysis of VEGFA protein expression levels after transfection with JUP siRNA in HCT8 cells. (F-H) Western blot analysis of VEGFA protein expression levels after transfection with POFUT2 overexpression plasmid and POFUT2 siRNA in HCT8 cells. Data are presented as mean ± standard deviation and were analyzed using T-test statistical analysis, with n = 3, **P < 0.01, ***P < 0.001, ns: not significant (P > 0.05).

Article Snippet: It was then incubated with AAL lectin (Vector Labs, B-1395-1) at 4 °C overnight.

Techniques: Expressing, Western Blot, Over Expression, Quantitative RT-PCR, Knockdown, Transfection, Plasmid Preparation, Standard Deviation

Journal: Frontiers in Immunology

Article Title: MGAT1 knockout in human dendritic cells enhance CD8 + T cell activation

doi: 10.3389/fimmu.2025.1588795

Figure Lengend Snippet: Successful and stable MGAT1 KO in MUTZ-3 precursor cells (PCs) eliminates cell surface complex and hybrid N-glycans. (A) Simplified overview of MGAT1 function in N-glycan processing. MGAT1 initiates the conversion of high-mannose glycans to hybrid and complex N-glycans by adding a GlcNAc to the core structure. In the absence of MGAT1 ( MGAT1 KO), this step is blocked, resulting in accumulation of high-mannose glycans. Created in BioRender. Blomberg, A. (2025) https://BioRender.com/oheioep (B) Chromatograms displaying Sanger sequencing results for genotyping of MUTZ-3 WT and MGAT1 KO pool. The gRNA target cleavage sites are marked by a vertical dashed red line. The initial sequencing (1st seq) and the subsequent sequencing (re-seq) were performed with 7 weeks of routine cell passaging in between. (C) Summary of Indel % and KO score for MGAT1 KO PCs at initial sequencing (1st seq) and the subsequent sequencing (re-seq) performed with 7 weeks of routine cell passaging in between. (D) Illustration of MUTZ-3 PC differentiation protocol. Created in BioRender. Blomberg, A. (2025) https://BioRender.com/v5iyr9n . (E) WT and MGAT1 KO PCs and iDCs were surface-labeled with various biotinylated lectins, followed by staining with AF488-streptavidin to assess the impact of MGAT1 KO on cell surface glycan structures. The histograms display representative data from a single experiment, while the accompanying bar graphs show the mean ± SD of lectin intensities relative to WT PCs from seven independent experiments. Cells from different passages and differentiations were used across the seven experiments. The dotted histograms in each plot serve as the negative control, stained exclusively with AF488-streptavidin. Statistical analysis was conducted using two-way ANOVA on matched datasets, followed by Sidak’s multiple comparisons test to assess differences between WT cells and MGAT1 KO cells. Statistical significance is denoted as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Glycan structures were created in BioRender. Blomberg, A. (2025) https://BioRender.com/h2ia4rl .

Article Snippet: Briefly, cells were stained with LIVE/DEADTM Fixable Yellow Dead Cell Stain to assess viability, before incubation with a selection of biotinylated lectins from Vector Laboratories: Aleuria aurantia lectin (AAL), Maackia amurensis lectin I and II (MAL-I and MAL-II), Sambucus nigra agglutinin (SNA), Phaseolus vulgaris lectin L (PHA-L) and Peanut agglutinin (PNA).

Techniques: Glycoproteomics, Sequencing, Passaging, Labeling, Staining, Negative Control

Journal: Journal of Translational Autoimmunity

Article Title: Afucosylated IgG in idiopathic nephrotic syndrome patients with anti-nephrin autoantibodies correlate with disease activity

doi: 10.1016/j.jtauto.2025.100307

Figure Lengend Snippet: Evaluation of IgG fucosylation in healthy donor samples using biotinylated lectins under competitive and enzymatic treatments. Panel A shows a schematic representation of N-glycans linked to Asn297 of IgG, depicting core (α1,6-linked) and antennary (α1,2-linked) fucosylation. Panels B and C show the levels of fucosylated IgG from serum of healthy donors, either untreated or preincubated with L-fucose or D-lactose to compete for binding to AAL (Panel B) or UEA-I (Panel C) or pretreated with PNGase F to enzymatically remove N-glycans (Panels B and C). These experiments were performed to confirm lectin specificity in the detection of IgG fucosylation under defined conditions.

Article Snippet: After five washes, 100 μL of Aleuria Aurantia Lectin (AAL; Vector Laboratories Cat# B1395-1) or biotinylated Ulex Europaeus Agglutinin I (UEA-I; Vector Laboratories Cat# B1065-2), both diluted 1:1000 in SuperT, were added and incubated ON at 4 °C.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Studying macromolecular composition in cell-cell interfaces using 3D membrane reconstitution systems

doi: 10.1101/2025.11.01.686034

Figure Lengend Snippet: Schematic representation of the lectins used (AAL, SNA, WGA, MAL-II) to target different glycosylation patterns in the T cell glycocalyx and their distribution at the T cell GUV contact. B-E| Representative confocal image of contact formation between GUVs decorated with CD2 with Jurkat T cells labelled with WGA (B), AAL (C), SNA (D) and MAL-II (E) (left). CD2-AF488 intensity and WGA-AF647, AAL-AF647, SNA-AF647 or MAL-II-AF647 intensities at the contact and outside were quantified and normalized (middle). Linearized fluorescence intensity profiles of the GUV and cell stained with the respective lectin in the confocal image with the contact highlighted in grey (right). Superplots show individual contacts as small symbols. Large symbols depict the mean of the individual biological replicates. Symbol corresponds to the individual biological replicate (n=3). Standard error of the mean is shown. Scale bar corresponds to 10 μm.

Article Snippet: We also used the proteins: CD43/ human leukosialin (His-Tag, CD3-H52H9, ACROBiosystems), MUC1-Alexa FluorTM 488 (kindly provided by Carolyn Shurer), MHC class I H-2D b presenting the LCMV-derived gp33 peptide (KAVYNFATM) ( ; ), MHC class I (HLA-A*02:01) presenting the tumour-associated antigen NY-ESO1-9V peptide (SLLMWITQV; from now on 9V) , MHC class I (HLA-A*02:01) presenting the NY-ESO1-3P9V peptide variant (SLPMWITQV; from now on 3P9V), P14 TCR , 1G4 TCR , Wheat Germ Agglutinin (WGA) Alexa FluorTM 647 Conjugate (ThermoFisherScientific), Aleuria Aurantia Lectin (AAL) (L-1390-2, Vector Labs), Sambucus Nigra-I-Agglutinin (SNA) (L-1300-5, Vector Labs), Maackia Amurensis-II Lectin (MAL-II) (L-1260-2, Vector Labs).

Techniques: Glycoproteomics, Fluorescence, Staining