goat polyclonal anti mouse p selectin antibody  (R&D Systems)

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

Journal: Nature Communications

Article Title: A drug-free cardiovascular stent functionalized with tailored collagen supports in-situ healing of vascular tissues

doi: 10.1038/s41467-024-44902-2

Figure Lengend Snippet: a Representative rhodamine-conjugated phalloidin (TRITC-Phalloidin, red) and 4′,6-diamidino-2-phenylindole (DAPI, blue) fluorescence images of HUVECs showing the (rhCol III/PDA-PEI) n coatings loaded with different amounts of rhCol III all encouraged HUVECs compared with control PLA group. Scale bars, 200 μm. Quantification of ( b , c ) cell number and ( d , e ) cell viability of HUVECs cultured on uncoated and (rhCol III/PDA-PEI) n -coated PLA ( n = 1, 2, and 4) sheets after 1, 3, 5, and 7 days of culture ( n = 5 independent samples). f Volcano plot showing differentially expressed genes in the (rhCol III/PDA-PEI) 2 group compared to the PLA control group. Downregulated and upregulated genes are colored blue and red, respectively, at significantly differentially expressed thresholds │log 2 FC│ > 1.2 and p < 0.05. g Gene Ontology (GO) analysis of differentially expressed genes in PLA versus (rhCol III/PDA-PEI) 2 . h Heatmap of the differentially expressed genes in the PLA and (rhCol III/PDA-PEI) 2 groups. i Circular visualization of the results of gene-annotation enrichment analysis. j Quantification of the expression of representative genes in PLA versus (rhCol III/PDA-PEI) 2 , validated by qRT-PCR arrays. The value in the PLA group was normalized to that in the (rhCol III/PDA-PEI) 2 group ( n = 3 independent samples). k Representative immunofluorescence staining of CCL5 (green), GATA3 (green), XBP1 (green), and CEACAM6 (green) of HUVECs on 3 days of culture. Scale bar, 50 µm. l Schematic diagram of the potential three signaling pathways involved in the regulation of HUVEC behavior induced by the (rhCol III/PDA-PEI) 2 coating, including PI3K/AKT, mTOR, and MAPK. Two-way ANOVA with Tukey’s multiple comparisons was used for the comparisons in ( b )–( e ) and ( j ). Two-sided Student’s t test with multiple testing corrections was used in ( f ) and ( g ). The data are presented as the mean ± SD ( p values < 0.05 were considered statistically significant).

Article Snippet: The primary antibodies used in this study included mouse monoclonal XBP1 (Cat. No.: sc-8015, Clone: F-4, Santa Cruz Biotechnology, USA, 1:50), mouse polyclonal CCL5 (Cat. No.: sc-365826, Clone: A-4, Santa Cruz Biotechnology, USA, 1:50), mouse monoclonal CEACAM6 (Cat. No.: sc-59899, Clone: 9A6, Santa Cruz Biotechnology, USA, 1:50), rabbit monoclonal GATA3 (Cat. No.: ab199428, Clone: EPR16651, Abcam, USA, 1:500), rabbit polyclonal F4/80 (Cat. No.: 29414-1-AP, Proteintech, China, 1:100), rabbit monoclonal CD68 (Cat. No.: ab283654, Clone: EPR23917-164, Abcam, USA, 1:100), rabbit polyclonal CD86 (Cat. No.: bs-1035R, Biosynthesis Biotechnology co., ltd, USA, 1:200), rabbit monoclonal CD206 (Cat. No.: 24595, Clone: E6T5J, Cell Signaling Technology, USA, 1:200), mouse monoclonal α-SMA (Cat. No.: ab7817, Clone: 1A4, Abcam, USA, 1:200), and rabbit monoclonal MMP2 (Cat. No.: 10373-2-AP, Clone: SB13a, Proteintech, USA, 1:200), mouse monoclonal CD31 (Cat. No.: ab9498, Clone: JC/70A, Abcam, USA, 1:200), and rabbit polyclonal eNOS (Cat. No.: ab5589, Abcam, USA, 1:100).

Techniques: Fluorescence, Control, Cell Culture, Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Protein-Protein interactions

Journal: Cells

Article Title: Increased Adipose Tissue Expression of IL-23 Associates with Inflammatory Markers in People with High LDL Cholesterol.

doi: 10.3390/cells11193072

Figure Lengend Snippet: Figure 4. Increased adipose CCL5 protein expression in people with high LDL-c and positively correlated with IL-23 protein. CCL5 protein expression was determined in people with low LDL-c (L-LDL-c) and high LDL-c (H-LDL-c), five each, using IHC. (A) The representative IHC images obtained from three independent determinations with similar results show the increased adipose tissue TNF-α expression (40× or inset ×100 magnification) in people with H-LDL-c (≥2.9 mmol/L) compared to those with L-LDL-c (<2.9 mmol/L). (B) Increased CCL5 protein expression is shown in the H-LDL-c group compared to the L-LDL-c group (p = 0.0053). Staining intensity shown as arbitrary units (AU). (C) IL-23 protein correlated with CCL5 protein in adipose tissue. p-values < 0.05 were considered as statistically significant. ** Highly significant.

Article Snippet: Briefly, adipose tissue sections were incubated with primary antibodies, i.e., 1:200 dilution of rabbit polyclonal anti-IL-23 antibody (Abcam® ab115759; Waltham, MA, USA), 1:200 dilution of rabbit polyclonal anti-TNF antibody (Novus Biologicals Centennial, CO, USA; NBP1–19532) and 1:500 dilution of rabbit polyclonal anti-CCL5 antibody (R&D Systems AF478) overnight at room temperature.

Techniques: Expressing, Staining