Addition of IL-2Rβ sub-domains to IL-7Rα does not alter the signal strength. (A) CTLL-2 cells (left) and their derivatives that express IL-7Rα (middle) or 7α/2β/2β (right) were washed and then cultured in the presence of various concentrations of IL-2 (○) or IL-7 (•) for 48 hours. [³H]thymidine was added during the last 4 hours of culture. Each point represents the mean value of triplicate samples. (B) Staining of CTLL-2 and its derivatives with anti–IL-7Rα antibodies (—) to determine the receptor surface expression level. Negative staining levels with isotype control are also shown (– – –). (C) Monitoring proliferation response upon cytokine stimulation by [³H]thymidine incorporation. CTLL-2 and its derivatives were subjected to proliferation assays in response to saturating dosage of IL-7 (100 ng/mL) as described in “Materials and methods, Proliferation assays.” There was no significant difference between IL-7Rα and 7α/7α/7α-2βA. Error bars represent standard deviation from triplicate samples. (D) Phosphorylation of signaling molecules in CTLL-2 transfectants in response to IL-7. Cells were stimulated with saturating dosage of IL-7 (100 ng/mL) for 10 minutes and then lysed. IL-2 stimulated and nonstimulated IL-7Rα⁺ CTLL-2 cells were applied as positive and negative controls, respectively. Immunoprecipitates and whole-cell lysates (WCL) were resolved by SDS-PAGE and transferred to membranes. The membranes were probed with anti-pTyr, and anti-pStat5 antibodies or probed with anti-Stat5 antibodies for loading controls. The amount of immunoprecipitated protein was determined by blotting with anti-Jak1, and anti-Jak3.