Human/Mouse/Rat RelA/NF kappaB p65 Antibody

Detection of RelA in HeLa Human Cell Line by Flow Cytometry.

HeLa human cervical epithelial carcinoma cell line was stained with Mouse Anti-Human/Mouse/Rat RelA/NF kappa B p65 Monoclonal Antibody (Catalog # MAB5078, filled histogram) or isotype control antibody (Catalog # MAB0041, open histogram), followed by Phycoerythrin-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # F0102B). To facilitate intracellular staining, cells were fixed with paraformadehyde and permeabilized with methanol.

Western Blot Shows Human RelA/NF kappaB p65 Specificity by Using Knockout Cell Line.

Western blot shows lysates of HeLa human cervical epithelial carcinoma parental cell line and RelA/NF kappa B p65 knockout HeLa cell line (KO). PVDF membrane was probed with 2 µg/mL of Mouse Anti-Human/Mouse/Rat RelA/NF kappa B p65 Monoclonal Antibody (Catalog # MAB5078) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF007). A specific band was detected for RelA/NF kappa B p65 at approximately 65 kDa (as indicated) in the parental HeLa cell line, but is not detectable in knockout HeLa cell line. GAPDH (Catalog # AF5718) is shown as a loading control. This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of Human RelA/NFkB p65 by Western Blot

Non-classical and intermediate monocytes express high levels of NF-kappa B (p65) and membrane-bound IL-1 alpha.a Western blot analysis of total p65 and GAPDH protein levels in the three monocyte subsets. b Quantification of Western blot data shown in (a): p65 protein level was normalized to GAPDH (loading control) and expressed as a fold change with respect to CL subset. The data represent the means ± SD; n = 3. c Relative levels of phosphorylated-p65 (p-p65), measured by flow cytometry. Each line represents one donor; n = 7. d IL-1 alpha secretion by the three monocyte subsets, measured by Luminex assay. The data represent the means ± SD; n = 3. e–f Relative expression levels of membrane-bound IL-1 alpha (e) and cytoplasmic IL-1 alpha (f), analyzed by flow cytometry. Each line represents one donor; n = 9. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. CL: classical, ITM: intermediate, NC: non-classical, MFI: median fluorescence intensity Image collected and cropped by CiteAb from the following publication (https://www.nature.com/articles/s41419-018-0327-1), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human RelA/NFkB p65 by Western Blot

Tumor‐derived EVs activate the NF kappaB pathway. Primary CD14+ monocytes were incubated with TEVs isolated from conditioned PCI‐1 supernatants. (A) Lysates were generated at different timepoints of incubation and tested for I kappaB and phosphorylated P‐I kappaB. Tubulin was used as a loading control. (B) Primary monocytes were incubated with complete PCI‐1 supernatant (SN), in TEV‐depleted SN (depl.) or with isolated TEV for 120 min, nuclei were isolated and the translocation of NF kappaB‐p65 was tested with an immunoblot. Actin was used as a loading control, and the cytoplasmic fraction (p65/cyto) was used to confirm the specificity of the translocation. (C) Monocytes were incubated as in (B), and the binding capacity of NF kappaB p50 to the DNA was quantified with a NF kappaB binding assay kit. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/29601673), licensed under a CC-BY license. Not internally tested by R&D Systems.