Human CD30 Ligand/TNFSF8 Antibody (116614)

Detection of CD30 Ligand/TNFSF8 by Flow Cytometry

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.

Detection of CD30 Ligand/TNFSF8 by Western Blot

Iso2 can block Iso1/CD30 interaction and anti-Iso1 antibody binding. (A) HEK cells were transfected with increasing quantities of untagged Iso1 (1NT) plasmid with a constant amount of either vector (vec) control or FLAG-tagged Iso2 (2FL). Lysates were used in a plate-based binding assay using CD30 recombinant protein as the capture and an Iso1-specific anti-CD30L antibody was used for detection. (B) HEK cells were transfected with 1NT with or without 2FL, and 2FL alone. The lysates were used in a plate-based binding assay using an anti-pan-CD30L antibody for capture and an Iso1-specific anti-CD30L antibody for detection. (C,D) HEK cells were transfected with two quantities of 1NT and a constant quantity of 2FL or vector (vec) control in 10 cm plates. Additionally, 10% of the lysates were set aside as the input whole-cell lysate fraction (C), and the remainder was incubated with CD30-Fc recombinant protein and protein A/G beads (D). Both were subject to SDS-PAGE and Western blot. 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.