mouse anti p selectin  (Proteintech)

Journal: Frontiers in Immunology

Article Title: Molecular determinants of STEC-HUS: from complement activation to microvascular thrombosis

doi: 10.3389/fimmu.2026.1749811

Figure Lengend Snippet: Prothrombotic effects of STEC-HUS serum are dependent on WPB exocytosis from endothelial cells. P-selectin (A) and vWF (B) expression on unstimulated HMEC-1 exposed to serum from patients with acute STEC-HUS (n = 5 for P-selectin experiments; n = 6 for vWF experiments), in the presence or in the absence of different complement inhibitors (sCR1, 150 µg/mL; factor B inhibitor, iptacopan,10 µM; eculizumab 100 µg/mL) or to a pool of control sera (normal human serum, NHS), run in parallel. Results are shown as pixel 2 /high-power field (HPF) of stained surface area. Data are mean ± SD. Circles represent single patients’ data. The addition of either sCR1, or iptacopan or eculizumab to patient’s serum significantly decrease both P-selectin and vWF expression induced by patient’s serum alone. *P < 0.05 vs STEC-HUS serum alone (ANOVA, followed by Tukey’s multiple comparisons test for data of P-selectin expression and by Holm-Šídák’s multiple comparisons test for data of vWF expression). (C) Endothelial surface area covered by thrombi on ADP-activated HMEC-1 exposed to serum from STEC-HUS patients collected during the acute phase of the disease and then perfused with whole blood. Before the experiments, HMEC-1 were left for 16 hours with medium added or not with the RalA inhibitor BQU57 (10 µM). Data are expressed as mean ± SD of percentages of serum-induced thrombus formation in respect to a pool of control sera (normal human serum, NHS), run in parallel in each experiment and set as 100% (n = 3 independent experiments). Circles indicate single patients’ data. Horizontal dashed lines indicate upper and lower limits of the normal range . *P < 0.05 (paired Student’s t test). (D) Representative confocal microscopy images (original magnification X200) of experiments of thrombus formation (green staining) relative to Figure 6C . Scale bar: 100 µm.

Article Snippet: Cells were washed again and treated with the following specific antibodies: FITC-conjugated rabbit anti-human C3c-complement (Dako, that recognizes C3c, part of C3 and C3b, 1:300 final dilution in Dapi 1 μg/mL); or rabbit anti-human complement C5b-9 complex (Calbiochem, 1:200 final dilution in PBS1X) followed by FITC-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, 1:50 final dilution in 1 μg/mL Dapi); or goat anti-human C4 (Abcam, 1:100 final dilution in PBS1X) followed by Cy3-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, 1:200 final dilution in Dapi 1 μg/mL); or FITC-conjugated anti-human IgG (Sigma Aldrich, 1:32 final dilution in 1 μg/mL Dapi); or mouse anti-human P-selectin (R&D System, 20 μg/mL final concentration in PBS1X), followed by Cy3-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, 1:60 final dilution in 1 μg/mL Dapi); or rabbit anti-human vWF (Dako, 10 μg/mL final concentration in PBS1X), followed by Cy3-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, 1:50 final dilution in 1 μg/mL Dapi).

Techniques: Expressing, Control, Staining, Confocal Microscopy

Journal: American journal of physiology. Cell physiology

Article Title: Endothelial cells retain inflammatory memory through chromatin remodeling in a two-hit model of infection-induced inflammation

doi: 10.1152/ajpcell.00567.2025

Figure Lengend Snippet: (A) Barplot of differentially expressed genes (DEGs) highlights substantial upregulation and downregulation specifically in CLP+ Sp mice across comparisons. (B) Heatmap of amplified genes demonstrates robust transcriptional activation in CLP+ Sp mice. (C) Transcription factor activity analysis identifies high predicted activation of STAT, IRF AND AP-1 and NF-kB complex members, supporting a coordinated transcriptional network driving inflammatory memory in the lung endothelium (DoRothEA, NES > 2). (D) Representative lung sections from sham+saline, sham+ Sp , CLP+saline, and CLP+ Sp mice were stained for P-selectin (red), Lectin (white) and nuclei (DAPI, blue) to assess endothelial activation at day 22 post-first hit. Scale bars, 100 μm. Representative of three independent cohorts, n=3-5.

Article Snippet: After three PBS washes, sections were permeabilized with 0.1% Triton X-100 in PBS (PBS-TX) for 15 min, then blocked in PBS-TX with 5% fetal bovine serum (FBS) for 1 h. Primary antibodies—P-Selectin (R&D Systems, Cat# AF737), E-Selectin (ABCAM, Cat# RM2070), VCAM1 (Cell Signaling, Cat# D8U5V), and Isolectin (Thermo Fisher Scientific, Cat# I32450 ), were applied in blocking buffer and incubated overnight at 4 °C or for 2 h at room temperature.

Techniques: Amplification, Activation Assay, Expressing, Infection, Activity Assay, Saline, Staining

Journal: bioRxiv

Article Title: P2Y12–P-Selectin Mediated Platelet Activation Drives Dengue-Associated Thrombocytopenia

doi: 10.1101/2025.09.08.674864

Figure Lengend Snippet: A129 mice (n = 6 per group), were subcutaneously inoculated in the plantar region with DENV-2 at a concentration of 2×10LJ PFU/animal or with saline solution (Mock group). Mice were monitored daily for weight loss and clinical score and euthanized on days 1, 3, and 5 post-infections, followed by blood collection for flow cytometry analysis. (B) Total platelets in peripheral blood: Selected from the DAPILJ CD41LJ cell population . (C) Activated platelets: Selected based on the DAPILJ CD41LJ CD62PLJ population . (D) Representative dot plots of platelet activation in peripheral blood. (E) Heatmap of plasma. Thrombopoietin (TPO), PF4 (CXCL4), CD62P soluble and NS1 concentrations in plasma were measured by ELISA and expressed as pg/mL of plasma. (F) Percentage of neutrophils: Identified in the CD45LJ CD11bLJ GR-1LJ F4/80LJ population, including aggregates with CD41LJ platelets. (G) Percentage of monocyte: Selected from the CD45LJ CD11bLJ GR-1LJ F4/80LJ population and aggregated with CD41LJ platelets. (H) Percentage of Dendritic cells: Identified in the CD45LJ CD11bLJ CD11cLJ MHCIILJ population, including aggregates with CD41LJ platelets. (I) Percentage of NK cells: Selected from the CD45LJ CD3LJ NKLJ population and aggregated with CD41LJ platelets. (J) Percentage of NKT cells: Identified in the CD45LJ CD3LJ NKLJ population and aggregated with CD41LJ platelets. (K) Percentage of CD4 D T lymphocytes: Selected from the CD45LJ CD3LJ CD4LJ population and aggregated with CD41LJ platelets. (L) Percentage of CD8 D T lymphocytes: Selected from the CD45LJ CD3LJ CD8LJ population and aggregated with CD41LJ platelets. (M) Percentage of B lymphocytes: Selected from the CD45LJ CD3LJ CD19LJ population and aggregated with CD41LJ platelets. Data presented in Figures D-E were obtained through logarithmic scale reading in the cytometer, excluding larger leukocytes. Figure E was generated by multiplying the total frequency by the total platelet count in peripheral blood. The symbols (*, **, ***, ****) indicate statistically significant differences between infected groups and the control group (Mock) (p < 0.05). Statistical analysis: Figures B and G: Kruskal-Walli’s test followed by Dunn’s post-test. Figures C-F and H-L: One-way ANOVA followed by Dunnett’s post-test. Figure A was created with Biorender. Batista, VL (2025).

Article Snippet: For P-selectin analysis, one hour before infection, mice received an intraperitoneal injection containing 30 μg of anti–P-selectin (CD62P) antibody (R&D Systems, MAB737) in 200 μL of saline as described previously[ ] .

Techniques: Concentration Assay, Saline, Flow Cytometry, Activation Assay, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Cytometry, Generated, Infection, Control

Journal: bioRxiv

Article Title: P2Y12–P-Selectin Mediated Platelet Activation Drives Dengue-Associated Thrombocytopenia

doi: 10.1101/2025.09.08.674864

Figure Lengend Snippet: A129 mice (n = 6 per group) received an intraperitoneal injection of 200 µL containing 30 µg/animal of murine anti-CD62P antibody (CD62P group), murine anti-IgG antibody (Isotype), with cell supernatant (Mock group) one hour before infection. One hour later, mice were subcutaneously inoculated via the intraplantar route with 30 µL of DENV-2 at a concentration of 2×10LJ PFU/animal diluted in saline or with cell culture supernatant (Mock group). After 48 hours of infection, mice received additional injections with the same concentrations of anti-CD62P antibody, IgG, or saline. Mice were monitored daily for weight loss and clinical signs, and three days post-infection, were euthanized for organ collection for flow cytometry and inflammatory parameter analyses. (B) Total Platelet Count in Peripheral Blood. Performed manually using a Neubauer chamber, with results expressed as 10³ platelets/mL of blood. (C) Percentage of CD62P-Positive Platelets in Peripheral Blood. (D) Representative dot plot of CD62P on the platelet surface. For the assessment of platelet activation, platelets were identified as DAPILJ CD41LJ, and within this population, CD62P expression was evaluated. Platelets were analyzed on a logarithmic scale to exclude larger cells . (E) Percentage of Neutrophils in Peripheral Blood. Identified within the CD45+ CD11b+ GR-1+ F4/80+ population, including aggregates with CD41+ platelets. (F) Percentage of Monocytes in Peripheral Blood. Identified within the CD45+ CD11b+ GR-1+ F4/80+ population, including aggregates with CD41+ platelets. (G) Plasma Inflammatory Mediator Heatmap. CCL2, CXCL1, and CCL5 quantification was performed by ELISA, with results expressed in pg/mL. (H) Percentage of Neutrophils in Lung. Identified within the CD45+ CD11b+ GR-1+ F4/80+ population, including aggregates with CD41+ platelets. (I) Percentage of Macrophages in the Lung. Identified within the CD45+ CD11b+ GR-1 - F4/80+ population, including aggregates with CD41+ platelets. (J) Percentage of Dendritic Cells in the Lung. Identified within the CD45+ CD11b+ CD11c+ MHCII+ population, including aggregates with CD41+ platelets . (K) Lung Inflammatory Mediator Heatmap. CCL2, CXCL1, and CCL5 quantification was performed by ELISA, with results expressed in pg/mg of lung tissue. (L) Lung Histopathological Score. (M) Representative H&E-Stained Lung Images. Statistical significance is indicated as follows: Symbols (*, **, ***, ****) denote significant differences between infected groups treated with Isotype compared to the Mock group (p < 0.05). The symbol (#) indicates significant differences between the anti-CD62P antibody-treated group and the Isotype group (p < 0.05). Data that passed the normality test were analyzed using ANOVA followed by Dunnett’s post hoc test, comparing the groups to the isotype control. Data that did not pass the normality test were analyzed using the Kruskal-Wallis test followed by Dunn’s post hoc test. Figure A was created with Biorender. Batista, VL (2025).

Article Snippet: For P-selectin analysis, one hour before infection, mice received an intraperitoneal injection containing 30 μg of anti–P-selectin (CD62P) antibody (R&D Systems, MAB737) in 200 μL of saline as described previously[ ] .

Techniques: Injection, Infection, Concentration Assay, Saline, Cell Culture, Flow Cytometry, Activation Assay, Expressing, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Staining, Control

Journal: bioRxiv

Article Title: P2Y12–P-Selectin Mediated Platelet Activation Drives Dengue-Associated Thrombocytopenia

doi: 10.1101/2025.09.08.674864

Figure Lengend Snippet: (A) Experimental design : A129 mice (n = 6 per group) were subcutaneously inoculated via the intraplantar route with 30 µL of DENV-2 (2×10LJ PFU/animal) diluted in saline or with cell supernatant (Mock group and Clop group). One hour after infection, mice were treated by gavage with an initial dose of 15 mg/kg of clopidogrel resuspended in 0.5% CMC, followed by a daily maintenance dose of 5 mg/kg under the same conditions. Mock and VEH groups received only the vehicle (0.5% CMC). The CLOP group (uninfected) received the same clopidogrel concentrations to assess drug toxicity . (B) Total platelets count in peripheral blood : Manually performed using a Neubauer chamber, with results expressed as 10³ platelets/mL of blood. (C) Percentage of CD62P-positive platelets in peripheral blood . ( D) Representative dot plot of the activated platelet population. (E) Percentage of Neutrophils in Peripheral Blood. Identified within the CD45+ CD11b+ GR-1+ F4/80+ population, including aggregates with CD41+ platelets. (F) Percentage of Monocytes in Peripheral Blood. Identified within the CD45+ CD11b+ GR-1+ F4/80+ population, including aggregates with CD41+ platelets. (G) Plasma Inflammatory Mediator Heatmap. CCL2, CXCL1, and CCL5 quantification were performed by ELISA, with results expressed in pg/mL. ( H) Percentage of Neutrophils in Lung. Identified within the CD45+ CD11b+ GR-1+ F4/80+ population, including aggregates with CD41+ platelets. (I) Percentage of Macrophages in the Lung. Identified within the CD45+ CD11b+ GR-1 - F4/80+ population, including aggregates with CD41+ platelets. (J) Percentage of Dendritic Cells in the Lung. Identified within the CD45+ CD11b+ CD11c+ MHCII+ population, including aggregates with CD41+ platelets. Symbols (*, **, ***, ****) denote significant differences between infected VEH and Mock groups (p < 0.05). Symbol # indicates significant differences between the clopidogrel-treated group and the infected VEH-treated group (p < 0.05). ANOVA with Dunnett’s post-test and Kruskal-Wallis with Dunn’s post-test were used, depending on the dataset.

Article Snippet: For P-selectin analysis, one hour before infection, mice received an intraperitoneal injection containing 30 μg of anti–P-selectin (CD62P) antibody (R&D Systems, MAB737) in 200 μL of saline as described previously[ ] .

Techniques: Saline, Infection, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Selective inhibition of ERO1α with M6766, a novel small-molecule inhibitor, prevents arterial thrombosis and ischemic stroke in mice

doi: 10.1016/j.ymthe.2025.07.033

Figure Lengend Snippet: (A–F) C57BL/6 mouse platelets were pretreated with vehicle (0.1% DMSO) or various concentrations of M6766 or EN460. (A and B) After incubation with 0.01 U/mL thrombin (Thr) or 0.025 μg/mL collagen-related peptide (CRP), P-selectin exposure and αIIbβ3 integrin activation were measured by flow cytometry. (C–F) After incubation with 0.015 U/mL thrombin or 0.05 μg/mL CRP, platelet aggregation and ATP secretion were measured using an aggregometer. (C and E) Representative traces of platelet aggregation. (G–I) Platelets in C57BL/6 mouse blood were labeled with DiOC6. After pretreatment with vehicle (0.1% DMSO) or 5 μM M6766 or EN460, blood was perfused under 50 dyne/cm 2 into a microfluidic chamber coated with type 1 collagen. Adherent and aggregated platelets were captured under an epifluorescence microscope. (J–N) Mouse platelets were pretreated with vehicle (0.1% DMSO) or various concentrations of M6766 or EN460. Ca 2+ release and influx were assessed in response to 0.02 U/mL thrombin, 0.5 μM A23187, or 5 μM thapsigargin, followed by the addition of 2 mM CaCl 2 . (M and N) The Ca 2+ signal was quantified by the area under the curve (AUC). (O) Biolayer interferometry was performed using a biotinylated STIM1 biosensor. After incubation of 2.5 μM ERO1α with 5 μM M6766, the specific interaction between STIM1 and ERO1α was measured by subtracting the nonspecific binding. The flow cytometric data are presented as the geometric mean fluorescence intensity (MFI). The data represent the mean ± SD ( n = 4–6 for A and B, n = 3 for C–F and O, and n = 4 for G–N). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. vehicle control after ANOVA and Dunnett’s test (A, B, and H–N) or Student’s t test (C–F and O).

Article Snippet: A goat polyclonal anti-mouse P-selectin antibody and mouse CCL5/RANTES DuoSet ELISA kit were purchased from R&D Systems (Minneapolis, MN).

Techniques: Incubation, Activation Assay, Flow Cytometry, Labeling, Microscopy, Binding Assay, Fluorescence, Control

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Selective inhibition of ERO1α with M6766, a novel small-molecule inhibitor, prevents arterial thrombosis and ischemic stroke in mice

doi: 10.1016/j.ymthe.2025.07.033

Figure Lengend Snippet: (A–J) P-selectin exposure, integrin activation, aggregation, and ATP secretion of WT control. (A–D) Ero1β-null or (E–J) Ero1α/β-null platelets were induced by various concentrations of thrombin (Thr) or CRP. (K–N) WT control or Ero1α/β-null platelets were pretreated with vehicle (0.1% DMSO) or various concentrations of M6766 or EN460. (K and L) After incubation of Ero1α/β-null platelets with 0.01 U/mL thrombin and 0.025 μ/mL CRP, P-selectin exposure and αIIbβ3 integrin activation were measured by flow cytometry. (M and N) After incubation of WT control or Ero1α/β-null platelets with 0.015 U/mL thrombin, platelet aggregation and ATP secretion were measured using an aggregometer. The data represent the mean ± SD ( n = 3 for A–D, G–J, M, and N and n = 5 for E, F, K, and L). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. vehicle control after Student’s t test (A–J) or ANOVA and either Dunnett’s test (K and L) or Tukey’s test (M and N).

Article Snippet: A goat polyclonal anti-mouse P-selectin antibody and mouse CCL5/RANTES DuoSet ELISA kit were purchased from R&D Systems (Minneapolis, MN).

Techniques: Activation Assay, Control, Incubation, Flow Cytometry

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Selective inhibition of ERO1α with M6766, a novel small-molecule inhibitor, prevents arterial thrombosis and ischemic stroke in mice

doi: 10.1016/j.ymthe.2025.07.033

Figure Lengend Snippet: (A and B) C57BL/6 mice were treated with intravenous injection of vehicle (1% DMSO in saline), M6766, or EN460 (0.3 μg/g body weight [BW]). Ten minutes later, blood was collected, and platelets were isolated. P-selectin exposure and αIIbβ3 integrin activation were assessed in flow cytometry. (C) Plasma concentrations of M6766 were analyzed by LC-MS/MS after intravenous injection of the compound (0.3 μg/g BW) into C57BL/6 mice and quantified by comparison with a standard curve of M6766 (mean ± SD, n = 3). (D and E) C57BL/6 mice were pretreated with intravenous injection of vehicle, M6766, or EN460 (0.3 μg/g BW), followed by injection of a DyLight 649-conjugated anti-CD42c antibody. Ten minutes later, intravital microscopy was performed to quantify the median integrated fluorescence intensities of an anti-CD42c antibody ( n = 30–32 arterioles in 5 mice per group). (D) Representative images. (E) Quantification of the antibody signal at various time points after laser injury. (F and G) C57BL/6 mice were pretreated with intravenous injection of vehicle, M6766, EN460 (0.3 μg/g BW), or eptifibatide (5 μg/g BW) 10 min before applying a (F) 7% or (G) 10% FeCl 3 -soaked filter paper to a carotid artery. The TTO was measured using a Doppler flow meter. (H and I) C57BL/6 mice were treated with intravenous injection of vehicle, M6766, EN460 (0.3 μg/g BW), or eptifibatide (5 μg/g BW). Ten minutes later, tail bleeding times and hemoglobin contents were measured after amputation of the tail tip. (J–M) C57BL/6 mice were subjected to transient middle cerebral artery occlusion for 1 h, followed by intravenous injection of vehicle, M6766, or EN460 (0.3 μg/g BW). Twenty-three hours later, neurological deficits were assessed by the Bederson score and grip strength test. Infarct volume was measured as described in . The data represent the mean ± SD ( n = 3 for A–C and n = 7 for M). The bar indicates the median ( n = 5 for E and n = 7–8 for F–I). * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. vehicle control after Student’s t test (A, B, and M) or Mann-Whitney U test (E–K).

Article Snippet: A goat polyclonal anti-mouse P-selectin antibody and mouse CCL5/RANTES DuoSet ELISA kit were purchased from R&D Systems (Minneapolis, MN).

Techniques: Injection, Saline, Isolation, Activation Assay, Flow Cytometry, Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy, Comparison, Intravital Microscopy, Fluorescence, Control, MANN-WHITNEY