Establishment of hIL5R overexpressing TF-1 cells. (A) Surface expression of hIL5R protein in TF-1, JYTF-1, and hIL5R transfectants TF1 and TF8 cells. The target cells were cultured in IL-5 for 24 hours and then subjected to flow cytometric analysis as described in Materials and Methods. The primary antibody was either a control mouse IgG (dashed line) or a mouse monoclonal anti-hIL5R antibody (solid line). The X-axis indicates the viable cell number and the Y-axis shows the fluorescence intensity. (B) Expression of the virus-derived IL5R mRNA in TF1 and TF8 cells. Total RNA was isolated from the indicated cell lines and 20 μg of RNA was analyzed by Northern blot hybridization. Probes used were cDNA-specific for the IL5R gene (upper panel) or for the glyceraldehyde-3–phosphate dehydrogenase (G3PDH) gene (lower panel), which were used as internal controls for the loading control. The positions of the endogenous full-length IL5R mRNA (IL5R) and the virus-encoded IL5R mRNA (v IL5R) are indicated at the right-hand side of the figure. (C) Distinct growth curve of TF-1, JYTF-1, TF1, and TF8 cells in IL-5. Cells were grown in the absence of cytokine (○) or in the presence of GM-CSF (□) or IL-5 (▵) at densities of 5 × 10⁵ cells/mL and 1 × 10⁵ cells/mL (GM-CSF and IL-5), respectively. Viable cell number was counted at 0 hours, 24 hours (day 1), and 48 hours (day 2) after seeding and was converted into the percentage of initial cell number (at day 0) as shown on the left-hand side of each plot. Each value is the average of four independent measurements.