Ligand–receptor binding analysis for IL-20R2302. (a) Both IL-20-AP and IL-24-AP fusion proteins could bind to mouse IL-20R2308 in vitro. 0.12 unit of either mIL-20R2308-AP or AP as a negative control were separated on a non-reducing SDS-PAGE and detected by either western blot analysis with an AP antibody or in situ affinity staining with IL-20-AP and mIL-24-AP fusion proteins after transferring to a PVDF membrane. (b) Cell surface staining assay for IL-20R2302 receptor binding. COS-E5 cells transfected with either mouse IL-20R1/IL-20R2308 or IL-20R1/IL-20R2302 heterodimeric receptors were stained with IL-20-AP and viewed under a light microscope (× 10 and × 40). COS-E5 cells transfected with pCDEF vector alone served as a negative control. Cells transfected with both IL-20 receptor heterodimers (IL-20R1/IL-20R2308 and IL-20R1/IL-20R2302) gave numerous positive cells with cell surface staining, confirming that the IL-20R2302 receptor is functional in ligand binding. The results shown were representative of at least three independent experiments.