Human Tie-2 Antibody

Detection of Human Tie‑2 by Western Blot. Western blot shows lysate of HUVEC human umbilical vein endothelial cells. PVDF membrane was probed with 1 µg/mL of Goat Anti-Human Tie-2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF313) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (HAF017). A specific band was detected for Tie-2 at approximately 150 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Tie‑2 in Human Placenta. Tie-2 was detected in immersion fixed paraffin-embedded sections of human placenta using Goat Anti-Human Tie-2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF313) at 10 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; CTS008) and counterstained with hematoxylin (blue). View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.

Detection of Human Tie‑2 by Simple Western^TM. Simple Western lane view shows lysates of HUVEC human umbilical vein endothelial cells, loaded at 0.2 mg/mL. A specific band was detected for Tie-2 at approximately 161 kDa (as indicated) using 10 µg/mL of Goat Anti-Human Tie-2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF313) followed by 1:50 dilution of HRP-conjugated Anti-Goat IgG Secondary Antibody (HAF109). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Detection of Human Tie-2 by Western Blot The activation and invasive potential of HSC are prevented by a Tie2 neutralizing antibody or by the inhibition of Akt/PI3k and MAPK signaling pathways.(A) alpha -SMA expression was assessed by western blotting (1A4, 1∶1000) in 20 µL of Laemmli lysates from HSC exposed during 24 h to different culture conditions (hepatic-derived CM, 0% FBS DMEM or 10% FBS DMEM) in presence (+) or absence (-) anti-Tie2 blocking antibody (AF313, 8 µg/ml). Quantitative analysis of alpha -SMA/tubulin bands in presence of neutralizing antibody is displayed in the graph as percentage of expression observed without anti-Tie2 (100%) for each experimental condition. Bars show mean +SD of 3 independent experiments. *p<0.05. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/25302785), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Tie-2 by Western Blot The activation and invasive potential of HSC are prevented by a Tie2 neutralizing antibody or by the inhibition of Akt/PI3k and MAPK signaling pathways. (C) The influence of different CM in presence or absence of LY294002 and PD98059 at 25 µmol/ml each (Akt/PI3k and MAPK inhibitors, respectively) on the expression of Tie2 (AF313, 1∶200) and alpha -SMA (1A4, 1∶1000) by HSC is illustrated. Respective western blots were analyzed in relation to tubulin to normalize total protein loading and the expression in presence of inhibitors was displayed as percentage of the expression without the respective compound (100%) in the same experimental conditions. Data from 3 experiments in duplicate are shown. *p<0.05. (D) Invasive potential of HSC (5×104 cells/100 µL) under the influence of LY294002 and PD98059 (25 µmol/ml both) at different experimental conditions (hepatic-derived CM, 0% FBS DMEM or 10% FBS DMEM) is shown by fluorescence images (400x). Graph depicts the mean +SD of average migrating HSC/field (5 microscope fields) per experiment (3 independent) of denoted HSC culture conditions. *p<0.05: statistical difference of presence versus absence of inhibitors. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/25302785), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Tie-2 by Western Blot Activation of HSC increases Tie2 expression.(A) Kinetics of Tie2 expression during in vitro activation of HSC. HSC, plated at 50,000 cells/cm2 density and grown in 2% DMEM during 24, 48, 72 and 96 hours, were lysed in Laemmli buffer and loaded in 7% acrylamide gels (20 µL of total protein extract per well). SDS-PAGE resolved proteins were transferred to nitrocellulose membranes and probed with respective antibodies against COL-I (H-197, 1∶2000), Tie2 (AF313, 1∶200) or tubulin (DM1A, 1∶5000). Quantitative analysis of Tie2 or COL-1 chemiluminescence in relation to tubulin (FUJIFILM Science Lab Image Gauge) is shown in respective graphs. *p<0.05 versus 24 h (mean +SD, 3 independent experiments). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/25302785), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Tie-2 by Western Blot Activation of HSC increases Tie2 expression. (D) HSC conditioned during different times with media from hepatic cells (Huh7 or HCV replicons) or 10% FBS DMEM. HSC, plated at same density in 0% FBS DMEM 24 h before, were exposed during 24, 48 or 72 h to 10% FBS DMEM or CM from hepatic cell lines and the expression of Tie2 (AF313, 1∶200), normalized with tubulin (DM1A, 1∶5000), was analyzed. Graph shows quantitative densitometric analysis of western blot bands (mean +SD of 3 independent experiments). #p<0.05: statistic differences versus Huh7 at same times; *p<0.05, same CM versus 24 h. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/25302785), licensed under a CC-BY license. Not internally tested by R&D Systems.