Human CD30 Ligand/TNFSF8 Antibody

Detection of CD30 Ligand/TNFSF8 in Human PBMCs by Flow Cytometry.

Human peripheral blood mononuclear cells (PBMCs) treated with 50 ng/mL PMA and 200 ng/mL Calcium Ionomycin overnight were stained with Mouse Anti-Human CD3e APC-conjugated Monoclonal Antibody (Catalog # FAB100A) and either (A) Mouse Anti-Human CD30 Ligand/TNFSF8 Monoclonal Antibody (Catalog # MAB1028) or (B) Mouse IgG_2BIsotype Control (Catalog # MAB004) followed by Allophycocyanin-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # F0101B). View our protocol for Staining Membrane-associated Proteins.

IL-6 Secretion Induced by CD30 Ligand/TNFSF8 and Neutralization by Human CD30 Ligand/TNFSF8 Antibody.

In the presence of a cross-linking antibody, Mouse polyHistidine Monoclonal Antibody (10 µg/mL, Catalog # MAB050), Recombinant Human CD30 Ligand/TNFSF8 (Catalog # 1028-CL) stimulates IL-6 secretion in the HDLM human Hodgkin’s lymphoma cell line in a dose-dependent manner (orange line), as measured by the Human IL-6 Quantikine ELISA Kit (Catalog # D6050). Under these conditions, IL-6 secretion elicited by Recombinant Human CD30 Ligand/TNFSF8 (1 µg/mL) is neutralized (green line) by increasing concentrations of Mouse Anti-Human CD30 Ligand/TNFSF8 Monoclonal Antibody (Catalog # MAB1028). The ND₅₀ is typically 1-4 µg/mL.

Detection of CD30 Ligand/TNFSF8 by Western Blot

Iso2 partially blocks Iso1-mediated stimulation of K299 cells. (A) HEK cells were transfected with either vector (vec) control or untagged Iso1 (1NT) with or without untagged Iso2 (2NT) or FLAG-tagged Iso2 (2FL). Transfected HEK cells were co-cultured with a K299-NFkB-Luc reporter clonal line. Two asterisks indicate p ≤ 0.01, three indicate p ≤ 0.001, and four indicate p ≤ 0.0001. (B,C) HEK cells were transfected with increasing quantities of 1NT plasmid with either vec or 2NT at a constant quantity, and then co-cultured with the K299-NFkB-Luc reporter line. Reporter activity (B) and IL-6 production (C) were measured. (D) HEK cells were transfected with either 1NT and vec or Iso2 and assessed for 1NT expression by flow cytometry. (E) Cells were transfected with increasing quantities of 1NT and either vec or 2FL, and the lysates were subject to SDS-PAGE and Western blot using a pan-CD30L antibody. Densitometry was used to quantify Iso1 bands, normalizing to GAPDH. Error bars for all are from standard deviations between technical replicates; representative experiment of ≥3 shown. Image collected and cropped by CiteAb from the following open publication (https://www.mdpi.com/1467-3045/46/3/172), licensed under a CC-BY license. Not internally tested by R&D Systems.