Figure 3.
IL-5 regulation of transmembrane IL-5Rα and sIL-5Rα expression. (a) TF1 cells (107/lane) were cytokine-starved for 48 hours and stimulated with 5 ng/ml human IL-5 for the indicated times. Whole-cell lysates were prepared and subjected to IP/IB with anti–IL-5Rα antibodies. The top arrow represents IL-5Rα (60 kDa), and the bottom arrow corresponds to sIL-5Rα (50 kDa). The multiple bands represent differential glycosylation (see below). (b) TF1 cells continuously cultured in IL-5 were immunoprecipitated with anti–IL-5Rα antibodies (lanes 1 and 2), and either left untreated (lane 1) or treated with PNGase F (lane 2) for 1–2 hours to remove N-linked glycosyl groups. Lanes 3 and 4 are controls for sIL-5Rα migration, using 20 ng of baculovirus-expressed, recombinant sIL-5Rα protein (brsIL-5Rα), which was either untreated (lane 3) or treated with PNGase F (lane 4) for 1–2 hours, separated by 12.5% SDS-PAGE, and immunoblotted with anti–IL-5Rα antibodies. Note how the multiple bands in lane 1 resolve to two distinct bands in lane 2 (PNGase F–treated). (c) TF1 cells (107/lane) were cytokine-starved for 48 hours and stimulated with 5 ng/ml IL-5 for the indicated times. Total RNA was prepared for each timepoint, and 30 μg of RNA/lane were analyzed with a probe specific to the extracellular domain of IL-5Rα (detects both isoforms). GAPDH was used as an internal control.