human itgb1  (Addgene inc)

Journal: Cancer research

Article Title: Targeting Interactions between Siglec-10 and α3β1 Integrin Enhances Macrophage-Mediated Phagocytosis of Pancreatic Cancer

doi: 10.1158/0008-5472.CAN-25-0977

Figure Lengend Snippet: (a) Experimental design for the pull-down of Siglec-10 ligands on PDAC cells. Recombinant Siglec-10 Fc (as well as a no-protein control or a Siglec-5 control) was allowed to bind its physiological ligands on the surface of PDAC cells, followed by an HRP-conjugated anti-Fc secondary antibody. In the presence of H₂O₂, HRP generated short-lived radicals that facilitated the transfer of biotin to proximal Siglec-10 ligands. Biotinylated Siglec-10 ligands were pulled down using streptavidin beads and identified by mass spectrometry. Created in BioRender. Saini, P. (2025) https://BioRender.com/lhnibjl . (b) A total of 4,044 proteins were identified, with enriched binding compared to a control using the anti-Fc antibody only without Siglec-10 protein. Of these, 110 proteins showed enrichment relative to a Siglec-5 control. Six proteins, CD47, CD59, CD73, ITGB6, ITGA3, and ITGB1, were significantly overexpressed in PAAD tissues compared to normal tissues in the TCGA dataset. (c) Response curves showing interactions between Siglec-10 and the six glycoproteins measured by surface plasmon resonance (SPR). Two concentrations (1000 nM, green; 100 nM, red) were tested for all glycoproteins, while ITGA3 was also tested at 300 nM (green) and 30 nM (red). (d) Binding of the SNA lectin (specific for sialic acid) to ITGA3 and ITGB1 recombinant glycoproteins was measured by a lectin array. Sialidase-treated glycoproteins (blue bars) showed significantly reduced binding compared to untreated glycoproteins (red bars). Unpaired t-tests. Means with SEM are shown. (e) SPR response curves comparing the binding of intact (sialylated) ITGA3 and desialylated ITGA3 to immobilized Siglec-10. (f) SPR response curves comparing the binding of intact (sialylated) ITGB1 and desialylated ITGB1 to immobilized Siglec-10.

Article Snippet: The following recombinant proteins were tested: CD47 (His tag, Acrobiosystems, Catalog# CD7-H5227), CD59 (His, Avitag, Acrobiosystems, Catalog# CD9-H82E3), NT5E/CD73 (His, Avitag, Acrobiosystems, Catalog# CD3-H82E3), ITGB6 (C-Myc/DDK, Origene, Catalog# TP317387), ITGA3 (C-Myc/DDK, Origene, Catalog# TP320975), and ITGB1 (C-Myc/DDK, Origene, Catalog# TP303818).

Techniques: Recombinant, Control, Generated, Mass Spectrometry, Binding Assay, SPR Assay

Journal: Cancer research

Article Title: Targeting Interactions between Siglec-10 and α3β1 Integrin Enhances Macrophage-Mediated Phagocytosis of Pancreatic Cancer

doi: 10.1158/0008-5472.CAN-25-0977

Figure Lengend Snippet: ( a–c ) Comparison of CD24 (a), ITGA3 (b), and ITGB1 (c) expression between normal tissues and PAAD tissues in the TCGA dataset. Unpaired t tests. ( d–f ) Survival analyses of pancreatic tumor patients in the TCGA dataset showing the correlation between CD24 (d), ITGA3 (e), and ITGB1 (f) expression and overall survival. ( g–i ) Expression of CD24 (g), ITGA3 (h), and ITGB1 (i) in the PDAC TME across different disease states. Kruskal–Wallis test with Dunn’s multiple comparisons correction. Means with SEM are shown.

Article Snippet: The following recombinant proteins were tested: CD47 (His tag, Acrobiosystems, Catalog# CD7-H5227), CD59 (His, Avitag, Acrobiosystems, Catalog# CD9-H82E3), NT5E/CD73 (His, Avitag, Acrobiosystems, Catalog# CD3-H82E3), ITGB6 (C-Myc/DDK, Origene, Catalog# TP317387), ITGA3 (C-Myc/DDK, Origene, Catalog# TP320975), and ITGB1 (C-Myc/DDK, Origene, Catalog# TP303818).

Techniques: Comparison, Expressing

Journal: Cancer research

Article Title: Targeting Interactions between Siglec-10 and α3β1 Integrin Enhances Macrophage-Mediated Phagocytosis of Pancreatic Cancer

doi: 10.1158/0008-5472.CAN-25-0977

Figure Lengend Snippet: ( a ) Schematic model illustrating Siglec-10-mediated suppression of macrophage phagocytosis. In the left panel, Siglec-10 on macrophages binds to glycan ligands on PDAC cells, including ITGA3, ITGB1, and CD24, triggering inhibitory signaling and suppressing phagocytosis. In the right panel, blocking Siglec-10 with an antibody prevents inhibitory signaling and enhances macrophage phagocytic capacity. ( b ) ELISA screening of recombinant antibodies from the top hybridoma clones for Siglec-10 binding. Binding to immobilized Siglec-10 (blue) and Siglec-5 (gray) proteins is shown. ( c ) Flow cytometric analysis of antibody selectivity, showing binding to CHO-K1 cells expressing Siglec-10 (blue) but not Siglec-5 (gray). ( d ) AUC analysis of in vitro phagocytosis assays screening various Siglec-10 antibody clones, along with commercially available anti-CD24 and anti-Siglec-10 antibodies, for their ability to enhance macrophage-mediated phagocytosis of AsPC-1 PDAC cells. Means with SEM are shown. ( e ) AUC analysis of the in vitro phagocytosis assay using the top-performing Siglec-10 antibody clone with macrophages differentiated from monocytes of four healthy donors. Statistical significance was determined using Friedman’s ANOVA test. Means with SEM are shown. ( f ) Time-course analysis of the in vitro phagocytosis assay comparing the top Siglec-10 blocking antibody clone (68A11A1, blue) with the isotype control (gray). Data represent n = 4 independent experiments. ( g ) ELISA-based binding analysis of the recombinant 68A11A1 antibody to immobilized recombinant Siglec-10 and Siglec-5 proteins across different dilutions. ( h ) Evaluation of the recombinant Siglec-10 antibody (clone 68A11A1) and anti-CD24 antibody in enhancing macrophage-mediated phagocytosis of multiple PDAC cell lines (AsPC-1, MIA PaCa-2, and PANC-1). Phagocytosis was normalized to the isotype control for each cell line and conducted using macrophages derived from monocytes of 5–8 healthy donors. Each symbol represents an individual donor; statistical significance was assessed using ratio paired t-tests compared to isotype control. Means with SEM are shown. ( i ) Triple co-culture assay involving cancer-associated fibroblasts (CAFs), PANC-1 PDAC cells, and monocyte-derived macrophages, showing phagocytosis kinetics, AUC quantification, and representative images. Statistical significance assessed using paired t-tests. ( j-k ) Flow cytometry analysis of CellTrace Violet (CTV)-labeled human CD8 + T cells co-cultured with human monocytes ± anti-Siglec-10 antibody in the presence of anti-CD3/CD28 beads for 5 days. ( j ) T cell proliferation; ( k ) granzyme B expression. ANOVA with post hoc comparisons.

Article Snippet: The following recombinant proteins were tested: CD47 (His tag, Acrobiosystems, Catalog# CD7-H5227), CD59 (His, Avitag, Acrobiosystems, Catalog# CD9-H82E3), NT5E/CD73 (His, Avitag, Acrobiosystems, Catalog# CD3-H82E3), ITGB6 (C-Myc/DDK, Origene, Catalog# TP317387), ITGA3 (C-Myc/DDK, Origene, Catalog# TP320975), and ITGB1 (C-Myc/DDK, Origene, Catalog# TP303818).

Techniques: Blocking Assay, Inhibition, Activation Assay, In Vitro, Glycoproteomics, Enzyme-linked Immunosorbent Assay, Recombinant, Clone Assay, Binding Assay, Expressing, Phagocytosis Assay, Control, Derivative Assay, Co-culture Assay, Flow Cytometry, Labeling, Cell Culture