Human IL-18 R alpha/IL-1 R5 Antibody

Detection of Human IL-18R alpha/IL-1 R5 by Western Blot

Soluble interleukin-18 receptor alpha complex is associated with interleukin-18 and the soluble form of the interleukin-18 receptor beta chain. (A) Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the serum interleukin (IL)-18 receptor alpha (IL-18R alpha) complex. Purified H44 monoclonal antibody (mAb) was coupled to a 5-mL HiTrap NHS-activated HP column (GE Healthcare). Pooled human blood serum (120 mL) was applied to this affinity column. The IL-18R alpha complex was eluted with elution buffer at a flow rate of 2 mL/minute. Every 1 mL of the elution buffer was collected into a test tube containing 50 mL of neutralization buffer (collected fractions were denoted in order as fractions 1, 2, 3 and so on). A 10-μL aliquot of every fraction (fractions 1 to 8) was treated with the same volume of sample buffer containing 4% SDS (Tris-Glycine SDS Sample Buffer (2×); Invitrogen). Electrophoresis was carried out in the presence of 0.1% SDS, and the gel was stained with Coomassie Brilliant Blue. (B) Western blots of the serum IL-18R alpha complex using an antihuman IL-18R alpha mAb are shown. Western blot analysis was performed using antihuman IL-18R alpha mAb 70625 (R&D Systems, Inc.). Lane 1: Western blot showing 1 μg of rhIL-18R alpha/Fc chimera protein (R&D Systems, Inc.). Lane 2: Western blot showing 5 μg of isolated serum IL-18R alpha complex. (C) Western blot showing serum IL-18R alpha complex using an antihuman IL-18 mAb. Western blot analysis was performed using antihuman IL-18 mAb clone 8 with 5 μg of isolated serum IL-18R alpha complex. (D) Western blot showing serum IL-18R alpha complex using an antihuman IL-18R beta mAb. Western blot analysis was performed using antihuman IL-18R beta mAb 132016 (R&D Systems, Inc.) with 5 μg of isolated serum IL-18R alpha complex. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/21435242), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human IL-18R alpha/IL-1 R5 by Flow Cytometry

Effect of TNF alpha treatment on KG-1 IL-18R and IL-1R surface expression.KG-1 cells (106/ml) were incubated with the indicated combinations of rIL-1 beta, rIL-18, rTNF alpha (10 ng/ml each) for 24 h. Cells were stained with IL-18R-PE and IL-1R-FITC followed by flow cytometry analysis. Results are representative of 3 separate experiments. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/19707556), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human IL-18R alpha/IL-1 R5 by Western Blot

The synergistic effect of TNF alpha and IL-1 beta/IL-18 on I kappaB zeta protein expression is partially suppressed with a TNF alpha-specific Ab, but completely blocked with IL-1 beta and/or IL-18 neutralization.KG-1 cells (106/ml) were stimulated with rIL-1 beta, rIL-18, rTNF alpha (10 ng/ml each) (A), or the indicated combinations of these cytokines (B and C) for the indicated time points. At selected time points, the cells were incubated with the recombinant proteins in the presence of IL-1ra (100 µg/ml), IL-18R Ab (10 µg/ml), TNF alpha Ab (10 µg/ml), or different combinations of these neutralizing agents. Protein-matched total cell extracts were analyzed by Western blotting using anti-serum against I kappaB zeta and actin Ab. Results are representative of at least 3 separate experiments. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/19707556), licensed under a CC-BY license. Not internally tested by R&D Systems.