Human TNF RI/TNFRSF1A Antibody

TNF RI/TNFRSF1A in Human Peripheral Blood Lymphocytes.

TNF RI/TNFRSF1A was detected in immersion fixed human peripheral blood lymphocytes using 5 µg/mL Human TNF RI/TNFRSF1A Antigen Affinity-purified Polyclonal Antibody (Catalog # AF225) for 3 hours at room temperature. Cells were stained (red) and counterstained (green). View our protocol for Fluorescent ICC Staining of Non-adherent Cells.

Detection of Human TNF RI/TNFRSF1A by Western Blot

NRP1 and TNFRs are in the same protein complexes. (A) HUVECs were infected with retrovirus expressing NRP1 or LacZ as a control, and then stimulated with TNF alpha (5 ng/mL, 15 min). Cell lysates were immunoprecipated with anti-TNFR1 or TNFR2 antibodies and immunoprecipates were analyzed for the presence of the TNFRs and NRP1 by Western blotting (left panels). Cell lysates were immunoblotted as load control (right panel). (B) HUVECs were infected lentivirus expressing control-shRNA and NRP1-shRNA, respectively, selected with puromycin for 48 h, stimulated with TNF alpha and then subjected to Western blotting. (C, D) HUVECs (5*104/well) were plated into a 24-well plate. Half of the wells were pre-incubated with an excess of “non-GpL” TNF alpha (2 μg/mL) to determine the non-specific binding of Gaussia. princeps luciferase TNF alpha (GpL-TNF alpha). Then all the wells were incubated with GpL-TNF alpha at different concentrations for 30 min at 37°C. The non-specific binding values of the “non-GpL” TNF alpha groups were subtracted from the corresponding total binding values. The data were analyzed by a non-linear regression to a single site using the GraphPad Prism 5 software. The experiments were performed in duplicates and independently repeated for 3 times. (E) HUVECs were stimulated with TNF alpha (5 ng/mL) for 15 min and then subjected to immunofluorescent staining with the indicated antibodies. Nuclei were counterstained with DAPI. Co-localization of NRP1 and TNFR1 in both plasmal membrane (arrow) and cytoplasm were observed. Scale bar, 10 μm. Image collected and cropped by CiteAb from the following open publication (https://www.frontiersin.org/articles/10.3389/fcell.2024.1210944/full), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human TNF RI/TNFRSF1A by Western Blot

NRP1 and TNFRs are in the same protein complexes. (A) HUVECs were infected with retrovirus expressing NRP1 or LacZ as a control, and then stimulated with TNF alpha (5 ng/mL, 15 min). Cell lysates were immunoprecipated with anti-TNFR1 or TNFR2 antibodies and immunoprecipates were analyzed for the presence of the TNFRs and NRP1 by Western blotting (left panels). Cell lysates were immunoblotted as load control (right panel). (B) HUVECs were infected lentivirus expressing control-shRNA and NRP1-shRNA, respectively, selected with puromycin for 48 h, stimulated with TNF alpha and then subjected to Western blotting. (C, D) HUVECs (5*104/well) were plated into a 24-well plate. Half of the wells were pre-incubated with an excess of “non-GpL” TNF alpha (2 μg/mL) to determine the non-specific binding of Gaussia. princeps luciferase TNF alpha (GpL-TNF alpha). Then all the wells were incubated with GpL-TNF alpha at different concentrations for 30 min at 37°C. The non-specific binding values of the “non-GpL” TNF alpha groups were subtracted from the corresponding total binding values. The data were analyzed by a non-linear regression to a single site using the GraphPad Prism 5 software. The experiments were performed in duplicates and independently repeated for 3 times. (E) HUVECs were stimulated with TNF alpha (5 ng/mL) for 15 min and then subjected to immunofluorescent staining with the indicated antibodies. Nuclei were counterstained with DAPI. Co-localization of NRP1 and TNFR1 in both plasmal membrane (arrow) and cytoplasm were observed. Scale bar, 10 μm. Image collected and cropped by CiteAb from the following open publication (https://www.frontiersin.org/articles/10.3389/fcell.2024.1210944/full), licensed under a CC-BY license. Not internally tested by R&D Systems.