integrin beta 1 function  (Developmental Studies Hybridoma Bank)

Journal: The American Journal of Pathology

Article Title: Inhibiting the Secreted RGDKGE Collagen Peptide Selectively Controls CD8 + T-Cell Migration on Denatured Collagen-IV and Enhances Their Accumulation in Tumors

doi: 10.1016/j.ajpath.2025.09.008

Figure Lengend Snippet: CD8 + T cells control tumor growth following targeting the RGDKGE collagen peptide. C57BL/6 mice were treated intraperitoneally with function-blocking anti–CD8α-depleting antibody (BP0061) or non-specific control antibodies. A: Example of flow cytometry analysis for CD8 + T cells from spleens of mice treated with non-specific control antibody (Ab Cont) or anti–CD8α-depleting antibody (Anti-CD8). B: Quantification of CD8 + T cells from spleens of mice treated with non-specific control antibody (Ab Cont) or anti–CD8α-depleting antibody (Anti-CD8). Data represent CD8 + T cells from four mice per group. C: Example of flow cytometry analysis for CD4 + T cells from spleens of mice treated with non-specific control antibody (Ab Cont) or anti–CD8α-depleting antibody (Anti-CD8). D: Quantification of CD4 + T cells from spleens of mice treated with either non-specific control antibody (Ab Cont) or anti–CD8α-depleting antibody (Anti-CD8). Data represent CD4 + T cells from four mice per group. E: Examples of CD8 + T cells (red) in B16F10 tumor section from either non-specific control antibody (Ab Cont) or anti–CD8α-depleting antibody (Anti-CD8) treated mice. F: Quantification of the mean CD8 + T-cell count from four different tumors from each treatment group using five to seven ×200 microscopic fields from each tumor. Data represent CD8 + T-cell counts from four different tumors from each treatment group. G: Quantification of B16F10 tumor size from control antibody-depleted (Ab Cont) or CD8-depleted (Anti-CD8) mice over 14 days. Data points represent tumor volume from four mice per condition. H: Quantification of B16F10 tumor size from control antibody-depleted mice treated with non-specific control antibody (Ab Cont) or anti-RGDKGE antibody [monoclonal antibody (Mab) XL313] over 14 days. Data points represent tumor volume from eight mice per condition. I: Quantification of B16F10 tumor size from CD8-depleted mice treated with non-specific control antibody (Ab Cont) or anti-RGDKGE antibody (Mab XL313) over 14 days. Data points represent tumor volume from seven to eight mice per condition. Data are given as means ± SEM ( B , D , and F – I ). ∗ P < 0.05, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001. Scale bars = 50 μm ( E ). SSC, side scatter.

Article Snippet: Mab XL313, functional blocking anti-CD8α (BP0061) used for depletion, and non-specific control antibodies were acquired from Bio X-Cell (Lebanon, NH).

Techniques: Control, Blocking Assay, Flow Cytometry, Cell Characterization

Journal: Frontiers in Immunology

Article Title: Acetylsalicylic acid disrupts SARS-CoV-2 spike protein glycosylation and selectively impairs binding to ACE2

doi: 10.3389/fimmu.2025.1706997

Figure Lengend Snippet: ASA inhibits S1-ACE2 binding in Vero cells. (A) Representative images and quantification of the binding of spike protein (RBD, red) on Vero cells following 24 h treatment with 20 nM S1 incubated overnight with control medium (S1) or increasing concentrations of ASA in control medium (S1+ASA 5 mg/L, S1+ASA 20 mg/L, and S1+ASA 50 mg/L). Cell treated with medium alone served as control. Nuclei were counterstained with DAPI (blue). Data are expressed as % of positive area per high power field at ×63 magnification (% area/field, n=3 per group). Scale bar 20 µm. (B) Schematic representation (upper panel, created with BioRender.com) and quantification (lower panel) of the ELISA assay used to evaluate the binding of ACE2 to S1 protein treated overnight with PBS 1X (S1) or with increasing concentrations of ASA in PBS 1X (S1+ASA 5 mg/L, S1+ASA 20 mg/L, and S1+ASA 50 mg/L). Data are expressed as percentage of relative binding compared to S1 (n=7 per group). (C) Schematic representation (upper panel, created with BioRender.com) and quantification (lower panel) of the ELISA assay used to evaluate the binding of an anti-S1 antibody to S1 protein treated overnight in PBS 1X (S1) or with increasing concentrations of ASA (S1+ASA 5 mg/L, S1+ASA 20 mg/L, and S1+ASA 50 mg/L). Data are expressed as percentage of relative binding compared to S1 (n=4 per group). (D) Representative phase-contrast images showing the cytopathic effects in Vero cells at 4 days following exposure to SARS-CoV-2-positive swab incubated for 1 h with control medium (SARS-CoV-2 positive swab) or with ASA 20 mg/L (SARS-CoV-2 positive swab + ASA 20 mg/L). Cell treated with ASA 20 mg/L served as control. Scale bar 50 µm. Results are shown as mean ± SEM and were analyzed with Tukey’s multiple comparison test. **p-value<0.01, and ***p-value<0.001 vs control; °°p-value<0.01, and °°°p-value<0.001 vs S1.

Article Snippet: At confluence, cells were exposed to control medium alone (EMEM supplemented with 2% FBS, 1X P/S) or with S1 at a concentration of 10 nM, 20 nM, and 50 nM in the presence of anti-ACE2 functional blocking antibody (α-ACE2, 2 μg/ml, Adipogen, AG-20A-0037PF) or the corresponding normal mouse IgG (IRR; Santa Cruz, sc-2025).

Techniques: Binding Assay, Incubation, Control, Enzyme-linked Immunosorbent Assay, Comparison, Significance Assay

Journal: Frontiers in Immunology

Article Title: Acetylsalicylic acid disrupts SARS-CoV-2 spike protein glycosylation and selectively impairs binding to ACE2

doi: 10.3389/fimmu.2025.1706997

Figure Lengend Snippet: Structural mapping and functional relevance of glycosylation sites on SARS-CoV-2 S1 protein upon ASA treatment. (A) Schematic representation of the SARS-CoV-2 Spike S1 protein, comprising a signal peptide (SP, aa 1–13), the N-terminal domain (NTD; aa 14–305) a short interdomain linker (IL, aa 306–318), the receptor binding domain (RBD, aa 319–541), and subdomain 1 (SD1, aa 542–591) and subdomain 2 (SD2, aa 592–686). Glycoproteomic profiling revealed 8 N-linked and 25 O-linked glycosylation sites across different domains of S1. Treatment of the S1 with ASA 20 mg/L selectively removed N-glycosylation at residue N61 and O-glycosylation at residues T250, S325, S494, T572, and T645 (indicated in red). (B) Representative Western blot and quantification of S1 acetylation after overnight incubation in PBS or with increasing concentrations of ASA (ASA 5 mg/L, ASA 20 mg/L, and ASA 50 mg/L). Acetyl-lysine levels were detected using a rabbit anti-acetyl-lysine antibody, while a mouse anti-RBD antibody was used as loading control for normalization. Results are shown as mean ± SEM and were analyzed with Tukey’s multiple comparison test. *p-value<0.05, and **p-value<0.01 vs PBS. (C) Western blot analysis of wild-type S1 (S1 WT), mutant S1 N61D, and the double mutant S1 N61D/S325A, confirming successful expression and comparable molecular weight of the produced recombinant proteins. (D) Quantification of ACE2 binding to recombinant S1 proteins using the ELISA-based functional assay described in Figure 1B . Data are expressed as a percentage of ACE2 binding relative to S1 WT (n=12 per group). Results are shown as mean ± SEM and were analyzed with Tukey’s multiple comparison test. ***p-value<0.001 vs S1 WT; °°°p-value<0.001 vs S1 N61D.

Article Snippet: At confluence, cells were exposed to control medium alone (EMEM supplemented with 2% FBS, 1X P/S) or with S1 at a concentration of 10 nM, 20 nM, and 50 nM in the presence of anti-ACE2 functional blocking antibody (α-ACE2, 2 μg/ml, Adipogen, AG-20A-0037PF) or the corresponding normal mouse IgG (IRR; Santa Cruz, sc-2025).

Techniques: Functional Assay, Glycoproteomics, Binding Assay, Residue, Western Blot, Incubation, Control, Comparison, Mutagenesis, Expressing, Molecular Weight, Produced, Recombinant, Enzyme-linked Immunosorbent Assay, Significance Assay