Human Cyclin E1 Antibody

Detection of Human Cyclin E1 by Western Blot. Western blot shows lysates of K562 human chronic myelogenous leukemia cell line and HEK293 human embryonic kidney cell line. Gels were loaded with 25 µg of cytoplasmic (Cyto) and 25 µg of nuclear (Nuc) extracts. PVDF membrane was probed with 1 µg/mL of Sheep Anti-Human Cyclin E1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF6810) followed by HRP-conjugated Anti-Sheep IgG Secondary Antibody (HAF016). Specific bands were detected for Cyclin E1 at approximately 50 - 60 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 8.

Cyclin E1 in Human Breast. Cyclin E1 was detected in immersion fixed paraffin-embedded sections of human breast using Sheep Anti-Human Cyclin E1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF6810) at 10 µg/mL overnight at 4 °C. Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (CTS013). Tissue was stained using the Anti-Sheep HRP-DAB Cell & Tissue Staining Kit (brown CTS019) and counterstained with hematoxylin (blue). Specific staining was localized to nuclei. View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.

Detection of Human Cyclin E1 by Simple Western^TM. Simple Western lane view shows lysates of HepG2 human hepatocellular carcinoma cell line, loaded at 0.2 mg/mL. A specific band was detected for Cyclin E1 at approximately 53 kDa (as indicated) using 10 µg/mL of Sheep Anti-Human Cyclin E1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF6810) followed by 1:50 dilution of HRP-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). This experiment was conducted under reducing conditions and using the 12‑230 kDa separation system.

Detection of Mouse Cyclin E1 by Western Blot Activation of Sox17 at onset of EC regeneration and Sox17-mediated Cyclin E1 expression. a qPCR analysis of gene expression in sorted CD31+ cells from mTmG-Scl mice before and after injury (12 mg/kg i.p.). Sox17, Vegfr2, and Ccne1 increased significantly at day 2 post-LPS compared to baseline. n = 3. Color scale: the fold change increases from red to white to green color. b Western blot analysis in fresh isolated ECs from wild-type mice and quantification c showed a 5-fold increase in Sox17 protein expression within 1 day following injury compared to baseline and followed by recovery within 3 days post-LPS. n = 3. d, e Western blot analysis of cultured HLMVECs in which Sox17 was overexpressed showed 2.5x fold increase in Cyclin E1 protein expression relative to control cells. n = 3. OE, overexpression. f Representation of the CCNE1 promoter region with Sox17 binding sites (circled numbers) and their sequences. g HLMVECs were retrovirally transduced with Sox17 or control plasmid for 3 days, and Ch-IP assay followed by qPCR was performed to amplify Sox17 binding sites in the CCNE1 promoter. n = 3. h 293T cells were transfected with a Sox17 overexpression plasmid containing CCNE1 luciferase reporter constructs. Luciferase values were normalized to Renilla luciferase control reporter values. A schematic representation of corresponding deletion constructs is presented in the right panel. n = 3 and duplicates per sample. **P < 0.01 and ***P < 0.001. Data are shown as mean ± SEM. Analysis was performed using one-way ANOVA for (c) and two-way ANOVA with Bonferroni post-tests for (e, g, h) Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/31073164), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse Cyclin E1 by Western Blot Overexpression of Sox17 in ECs induces EC proliferation and regeneration. Mixture of 50 μg plasmid with 100 μl liposomes was injected i.v. 3 h after LPS challenge (12 mg/dose i.p.) in wild-type mice. This plasmid has a Flag-tag added to the N-terminus of Sox17 protein coding region and expression is under the regulation of a mouse Cdh5 promoter. a Confocal microscopy of flag staining with CD31 and DAPI co-staining for nuclei in lung cryo-sections from mice receiving a control vector or a Sox17-construct to over-express Sox17. Scale bar = 50 μm (original panel) and 20 μm (enlarged panel). n = 6. OE, overexpression. b Co-localization coefficient for the fraction of Flag in CD31+ cells assesses the transgene expression in the endothelium. The Pearson correlation coefficient is significantly increased in Sox17-overexpressing mice compared to control mice. n = 6. c Western blot analysis and its quantification d showed a significant increase in the flag and Cyclin E1 expression in the pulmonary endothelial cells of mice with 3 days of Sox17 overexpression compared to vector mice. n = 3. e Quantification of BrdU+ nuclei in each field of 425 μm2 area in lung cryo-sections from vector-overexpressing and Sox17-overexpressing mice. n = 5 per group and 6 technical replicates per sample. Slides are co-stained with CD31-AF594, BrdU-AF488, and DAPI. Both groups show increased BrdU+ ECs at day 3 post-LPS as compared to baseline and the response was significantly greater in mice in which ECs overexpressed Sox 17. f Lung transvascular albumin permeability pre-LPS and post-LPS challenge in mice overexpressing endothelial Sox17 and control mice. n = 5. Mice overexpressing Sox17 in ECs showed significantly reduced vascular leakiness post-LPS when compared to control mice. g Survival curve of LPS challenge in control mice and mice over-expressing Sox17 in the endothelium. n = 11 per group. At this lethal dose of LPS (20 mg/kg), the death rate for control mice is 60% while for Sox17-overexpressed mice is 10%. h Model. LPS induces tissue hypoxia due to local oxygen depletion by infiltrating activated neutrophils, thereby stabilizing HIF-1 alpha resulting in upregulation Sox17 expression and Sox17 mediated expression of Cyclin E1. This activates cell cycle re-entry and EC proliferation, and restoration of endothelial integrity. **P < 0.01 and ***P < 0.001. Data are shown as mean ± SEM. Analysis was performed using two-way ANOVA with Bonferroni post-tests for (d–f) and Log-rank (Mantel-Cox) test for (g) Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/31073164), licensed under a CC-BY license. Not internally tested by R&D Systems.