Relationship between receptor binding and immunologic signal.

Approximately 500 mL of supernatant conditioned by the human leukemic cell lines were subjected to a GM-CSF ligand affinity column and eluted fractions were volume reduced in centrifugal filtration cartridges. Panel A: 25 μL of each of the volume reduced fractions was used in¹²⁵I-GM-CSF solution phase binding assays in the absence or presence of a large excess of unlabeled GM-CSF. Specifically bound radioactivity (spec bound, cpm) was calculated by subtracting the precipitated radioactivity in the absence of unlabeled GM-CSF from that in the presence of unlabeled GM-CSF. Bars represent the mean and SEM of duplicate experiments. Panel B: 30 μL of each of the volume reduced fractions was subjected to 10.5% SDS PAGE under reducing conditions and transferred to PVDF membrane. Immunoblotting was performed with anti-GMRα antibody 8G6. Positive control (+ve) was ligand affinity purified recombinant solGMRα. Shown are representative results for the U937 cell line.