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. 2001 Dec 15;108(12):1797–1806. doi: 10.1172/JCI13877

Figure 6.

Figure 6

IL-5 stimulation results in cellular refractoriness to other βc-engaging cytokines. (a) TF1 cells that had been cytokine-starved for 24 hours were unstimulated (lane 1), or were stimulated with 20 ng/ml IL-5 for 5 minutes (lane 2) or 1 hour (lane 3). After 1 hour of incubation in IL-5, cells in lane 4 were harvested, washed once in 1× PBS (to remove IL-5), and restimulated with 20 ng/ml IL-3 for 5 minutes. As a positive control (lane 5), cells were stimulated only with 20 ng/ml IL-3 for 5 minutes. Whole-cell lysates were prepared and immunoprecipitated with anti-βc monoclonal antibody and immunoblotted with monoclonal antibody 4G10 (upper panel). The membrane was stripped and reprobed with anti-βc polyclonal antibodies (bottom panel). The migration of βc and βIP are indicated. (b) Same as in a, except that cells were pretreated with 20 ng/ml GM-CSF for 5 minutes or 1 hour, washed once in 1× PBS (to remove GM-CSF), and restimulated with 20 ng/ml IL-5 for 5 minutes. As a positive control (lane 5), cells were stimulated with 20 ng/ml IL-5 for 5 minutes only.