Figure 2. Competition binding of ¹²⁵I-ANP in HEK-293 cells expressing wild-type or GDAY/AAAA mutant receptors.

Confluent HEK-293 cells in 6 cm² dishes were incubated in 2 ml of assay medium with 1 nM ¹²⁵I-ANP and increasing concentrations of unlabelled ANP at 4 °C for 1 h. Cells were washed four times, each with 2 ml of assay medium to remove the unbound radioligand, and then dissolved in 1 M NaOH as described in the Materials and methods section. Specific ¹²⁵I-ANP radioactivity was determined in the solubilized cell extract. The binding curves are derived from the specific binding data from at least three separate experiments. The results are expressed as percentage maximum of bound/free hormone. The results represent the means±S.E.M. for three independent experiments with triplicate dishes in each replicate.