Human E-Selectin/CD62E Antibody

Detection of E-Selectin/CD62E in HUVECs by Flow Cytometry. Human umbilical cord endothelial cells (HUVECs) were cultured for 6 hours in the presence of 25 ng/mL of rhTNF-alpha (210-TA, filled histogram) or rested (open histogram) and stained with Mouse anti-human E-selectin/CD62E (BBA16) followed by Goat anti-Mouse APC-conjugated secondary antibody (F0101B). Gates were set based on isotype control (MAB002, data not shown). Staining was performed using our Staining Membrane-associated Proteins protocol.

E-Selectin/CD62E in HUVEC Human Cells. E-Selectin/CD62E was detected in immersion fixed HUVEC human umbilical vein endothelial cells activated with TNF-a (Catalog # 210-TA-010) using Mouse Anti-Human E-Selectin/ CD62E Monoclonal Antibody (Catalog # BBA16) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (red; Catalog # NL007) and counterstained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

E‑Selectin/CD62E in Human Brain. E-Selectin/CD62E was detected in immersion fixed paraffin-embedded sections of human brain (occipital cortex) using 10 µg/mL Mouse Anti-Human E-Selectin/CD62E Monoclonal Antibody (Catalog # BBA16) overnight at 4 °C. Before incubation with the primary antibody tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (Catalog # CTS013). Tissue was stained with the Anti-Mouse HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS002) and counterstained with hematoxylin (blue). View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.

Detection of E-Selectin/CD62E by Western Blot Induction of MIR181B expression by methotrexate (MTX) or adenosine is adenosine receptor A3 (ADORA3) dependent.(A) Knockdown for adenosine receptors A1, A2A, A2B, and A3 in HUVECs was performed to analyze MIR181B expression. three biological replicates. One-way ANOVA. (B) MIR181B expression in HUVECs transfected with Ctl-siRNA or ADORA3 siRNA after treatment with MTX (10 µM) or Ad (50 µM) or (C) treatment with TNF-alpha (10 ng/ml) alone or in combination MTX (10 µM) or Ad (50 µM). Three biological replicates. One-way ANOVA and Unpaired two-tailed Student t test. (D) Western blot analyses of VCAM-1, ICAM-1, and E-Selectin expression in HUVECs transfected with Ctl-siRNA or ADORA3 siRNA in the presence of TNF-alpha (10 ng/ml) in combination with either MTX (10 µM) or Ad (50 µM). Three biological replicates. Unpaired two-tailed Student t test. (E) in the presence of miRNA negative control (NS-m) or MIR181B mimics (181b-m) stimulated with TNF-alpha (10 ng/ml) or in combination with MTX (10 µM) or Ad (50 µM). Please see Figure 2—source data 1. Three biological replicates. Unpaired two-tailed Student t test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. n.s. indicated non significance. All values represent mean ± SEM.Figure 2—source data 1.Knockdown efficiency for adenosine receptor siRNAs.mRNA, and protein expression analysis for (A, B) ADORA3 siRNA, (C, D) ADORA1 siRNA, (E, F) ADORA2A siRNA and (G, H) ADORA2B siRNA compared to control siRNA in HUVECs transfected for 36 hr. (A–H), n = 3. ***p<0.001. All values represent mean ± SEM. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/33416495), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of E-Selectin/CD62E by Western Blot Methotrexate (MTX) and Ad repress TNF-alpha -induced pro-inflammatory genes without affecting primary MIR181A-1 and MIR181B-1 expression.(A) Western blot analysis of VCAM-1, ICAM-1, and E-Selectin in HUVECs treated with or without MTX (10 µM) or Ad (50 µM), after stimulation of TNF-alpha (10 ng/ml) for 8 hr. Quantification of n = 3 independent experiments. (B) Real-time qPCR analysis of VCAM-1, ICAM-1, and E-Selectin in HUVECs treated with or without MTX (10 µM) or Ad (50 µM), after treatment with TNF-alpha (10 ng/ml) for 4 hr. Real-time qPCR analysis of (C) primary transcript of MIR181A1 or (D) primary transcript of MIR181B-1 in HUVECs treated with or without MTX (10 µM) or Ad (50 µM), after treatment with TNF-alpha (10 ng/ml) for 4 hr. (A–D), n = 3–6. *p<0.05; **p<0.01; ***p<0.001; ***p<0.0001. All values represent mean ± SEM. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/33416495), licensed under a CC-BY license. Not internally tested by R&D Systems.