biotinylated ricinus communis agglutinin i  (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) 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: Frontiers in Immunology

Article Title: CNS-compartmentalized IgG aggregates and glycosylation in multiple sclerosis contribute to oligoclonal bands and neuronal cytotoxicity

doi: 10.3389/fimmu.2026.1689835

Figure Lengend Snippet: IgG1 is the dominant component of OCBs in MS CSF and coexists with IgG3, kappa, and lambda chains. (A) MS CSF was focused by IEF, followed by immunoblotting with anti-IgG1, anti-IgG3, anti-kappa, and anti-lambda antibodies, followed by incubation with HRP-secondary antibodies and color detection. Overlapping OCB bands were observed in IgG1, IgG3, kappa, and lambda chains (arrows). Two representative MS CSF blots are shown. (B) IgG1 depletion abolishes all OCBs, confirming its major role. MS CSF was incubated with biotinylated antihuman IgG1 antibody, followed by depletion with M-280 streptavidin-conjugated Dynabeads to remove all IgG1 antibodies. The IgG1-depleted MS CSF IEF blot was probed with anti-IgG (H + L). The original (neat) CSF serves as a control.

Article Snippet: To detect IgG1 galactosylation, biotinylated Ricinus communis agglutinin I (RCA) (1 mg/ml in TBS; No. B-1085-1, Vector Labs) was used.

Techniques: Western Blot, Incubation, Control