Journal: The Journal of Biological Chemistry
Article Title: Endothelial Cell Activation Is Regulated by Epidermal Growth Factor-like Domain 7 (Egfl7) during Inflammation *
Figure Lengend Snippet: Egfl7 is repressed in endothelial cells under inflammatory conditions in vivo . A , left panel , in situ hybridization detection of Egfl7 transcripts in endothelial cell nuclei of adult mouse lungs ( blue staining , arrows ). Right panel and inset , CD31 immunostaining ( brown , arrows ) and hematoxylin counterstaining of a parallel section of the same area. Bar , 25 μm. B , expression levels of CD31 and Egfl7 transcripts in CD31 − cells ( white bars ) and CD31 + cells ( black bars ) isolated from mouse lungs using immunoaffinity and measured by duplex RT-qPCR using a mouse CD31-FAM or a mouse Egfl7-FAM TaqMan probe mixed with a mouse β-actin-VIC probe (see “Experimental Procedures”). The results are plotted as quantities relative to CD31 − controls values set to 1. RQ , relative quantities. C , LPS (5 mg/kg, +) or LPS-free PBS (−) was instilled in mice nostrils, and animals were sacrificed at the onset of treatment (0 h) or after 10 or 24 h; the lungs were dissected and processed for total RNA isolation. Expression levels of ICAM-1, VCAM-1, E-selectin, and Egfl7 were measured by duplex RT-qPCR using the indicated FAM-labeled TaqMan probe for the mouse transcript of interest and a mouse β-actin-VIC-labeled TaqMan probe and expressed as 2 −ΔΔ C T quantities relative to t = 0 h values set to 1. *, p
Article Snippet: All qPCR were performed in duplex PCR mixing cDNA with both the TaqMan FAM-labeled probe of the tested gene (Life Technologies) and a β-actin- or a β2-microglobulin-VIC-labeled probe and processed for qPCR in a StepOne machine.
Techniques: In Vivo, In Situ Hybridization, Staining, Immunostaining, Expressing, Isolation, Quantitative RT-PCR, Mouse Assay, Labeling