biotinylated aleuria aurantia lectin  (Vector Laboratories)

Journal: bioRxiv

Article Title: Differential TIM-3 glycosylation enables specific dual targeting CAR-T therapy in acute myeloid leukemia

doi: 10.64898/2026.04.22.719217

Figure Lengend Snippet: (A) TIM-3 expression on KASUMI-3 cells, primary AML blasts, and healthy immune subsets (CIK cells, monocytes, NK cells) assessed by flow cytometry using QuantiBRITE beads. REH (ALL cell line) served as negative control. (B) Short-term killing assay of TIM-3.CAR-CIK cells against CIK (n = 11) or KASUMI-3 (n = 8) cells compared with NT cells. Target cell lysis was evaluated by flow cytometry (E:T 5:1). (C) Short-term killing assay of TIM-3.CAR-CIK cells against monocytes (n = 8) or NK cells (n = 8) compared with NT (E:T 5:1). KASUMI-3 (n = 4) were included as positive control. (D) Immunoblot analysis of TIM-3 in lysates from monocytes, CIK cells, and KASUMI-3 cells following enzymatic treatment with PNGase F or broad neuraminidase, probed with a commercial anti–TIM-3 antibody (TIM-3-cmAb). GAPDH, loading control. Glycan symbols follow SNFG. (E) TIM-3 immunoprecipitates from monocytes, CIK cells, and KASUMI-3 cells treated with PNGase F or O- glycosidase and analyzed by immunoblot with TIM-3-cmAb and lectin far-western with Aleuria aurantia lectin (AAL; fucosylated epitopes). TGX stain-free total protein signal is shown as a loading/normalization control. (F) KASUMI-3 cells treated with vehicle (mock) or the fucosylation inhibitor 2F-peracetyl-fucose (SGN-2FF), followed by PNGase F or neuraminidase treatment and immunoblot/lectin probing with TIM-3-cmAb and AAL. See also Figure S3A . (G) Short-term killing assay of TIM-3.CAR-CIK cells against untreated or SGN-2FF-treated KASUMI-3 cells at various E:T ratios (5:1, 1:1, 0.5:1, 0.25:1 and 0.125:1, n = 8). (H) Affinity kinetics (left) and binding avidity at 1000 pN force (right) of TIM-3.CAR-CIK cells to untreated or defucosylated KASUMI-3 by LUMICKS analysis (n = 6). Immunoblot experiments (D-F) were repeated in three independent biological replicates with similar results. Data are presented as individual values and mean ± SD. Statistical significance was determined with repeated-measures two-way ANOVA with Bonferroni’s post hoc test (B, C) or using paired t test (G, H). ns, not significant; *p = 0.01, **p < 0.001, ***p = 0.0001 and ****p < 0.0001. Illustrations were created with Biorender.com. See also Figure S3 for loading-matched TIM-3 immunoprecipitation controls.

Article Snippet: Membranes were probed with anti-human TIM-3 antibody (TIM-3-cmAb) (1:250; R&D Systems, MAB23652), a recombinant monoclonal antibody derived from the TIM-3.CAR scFv (TIM-3 scFv-mAb) (1:500; GENEWIZ), biotinylated Aleuria aurantia lectin (AAL; 1:3000; Vector Laboratories, B-1395-1), and biotinylated Ricinus communis agglutinin I (RCA I; 1:3000; Vector Laboratories, B-1085-1).

Techniques: Expressing, Flow Cytometry, Negative Control, Lysis, Positive Control, Western Blot, Control, Glycoproteomics, Staining, Binding Assay, Immunoprecipitation

Journal: bioRxiv

Article Title: Differential TIM-3 glycosylation enables specific dual targeting CAR-T therapy in acute myeloid leukemia

doi: 10.64898/2026.04.22.719217

Figure Lengend Snippet: (A) Immunoblot profiling of TIM-3 glycoforms in monocytes, CIK cells, and KASUMI-3 lysates using a recombinant scFv-derived monoclonal antibody (TIM-3scFv-mAb) following enzymatic treatment with PNGase F or broad neuraminidase. GAPDH, loading control. (B) TIM-3 immunoprecipitates from healthy monocytes, KASUMI-3 cells, and primary AML blasts treated with neuraminidase and/or PNGase F and analyzed by lectin and antibody probing: Ricinus communis agglutinin I (RCA-I; terminal β-galactose/LacNAc motifs), CA19-9 (sialyl-Lewis A), CSLEX1 (sialyl-Lewis X), and TIM-3scFv-mAb. See also Figure S3B . (C) High-resolution immunoblot of TIM-3 species detected by TIM-3scFv-mAb in CIK cells, primary AML blasts, and KASUMI-3 cells. GAPDH, loading control. See also Figure S3C . (D) RT-qPCR expression profiling of glycosyltransferases (FUT7, FUT8, ST3GAL3, ST3GAL4, ST3GAL6) in monocytes, KASUMI-3 cells, and primary AML blasts. Data are plotted as fold-change relative to monocytes and normalized to 18S RNA; individual points denote biological samples where applicable. (E) Schematic model summarizing a glycoform-biased recognition framework in which AML-associated remodeling of TIM-3 N -glycans contributes to preferential TIM-3.CAR recognition of AML-enriched TIM-3 glycoforms. Representative N -glycan structures are proposed for TIM-3 in AML blasts, monocytes and CIK cells based on enzymatic perturbation and lectin/antibody probing. Sugar moieties drawn with dashed outlines indicate features not directly resolved/assigned. Glycan symbols follow SNFG. Immunoblot and lectin/antibody blot experiments (A-C) were repeated in three independent biological replicates with similar results. Illustrations were created with Biorender.com. See also Figure S3 for additional lectin/antibody probing of TIM-3 glycoforms and terminal galactose exposure.

Article Snippet: Membranes were probed with anti-human TIM-3 antibody (TIM-3-cmAb) (1:250; R&D Systems, MAB23652), a recombinant monoclonal antibody derived from the TIM-3.CAR scFv (TIM-3 scFv-mAb) (1:500; GENEWIZ), biotinylated Aleuria aurantia lectin (AAL; 1:3000; Vector Laboratories, B-1395-1), and biotinylated Ricinus communis agglutinin I (RCA I; 1:3000; Vector Laboratories, B-1085-1).

Techniques: Western Blot, Recombinant, Derivative Assay, Control, Quantitative RT-PCR, Expressing, Glycoproteomics

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