Figure 6
Analysis for the mutation of Arg173 to Ala residue of the recombinant human IP receptor. A). Western blot analysis. Fifty micrograms of COS-7 cells transfected with wild-type (WT) or a mutant IP receptor (R173A) cDNA was subjected to SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was probed with rabbit anti-IP peptide antibody. (B) The ligand binding activities of wild-type and mutant TP receptor. 300 μg of the protein prepared from the COS-7 cells transfected with cDNA of the wild-type (WT) or the R173A mutant was incubated with 4 nM [3H]-iloprost (30,000 cpm) in the absence or presence of unlabeled iloprost (1 μM) in a reaction volume of 100 μl. After 1 h incubation, the reaction was stopped and the binding activity of the recombinant IP receptor was measured as described in the methods. The binding activity of wild-type receptor was considered as 100% (2,000 cpm).