American Society of Hematology

Figure 1

$KL stimulation of Jurkat-K7 cells results in the activation of IL-7R, Jak3, and Stat5. (A) γc and IL-7Rα are tyrosine phosphorylated by and associated with Kit after KL stimulation and γc-associated Jak3 is tyrosine phosphorylated in response to KL stimulation. Jurkat-K7 cells were stimulated with KL for the times indicated and γc and IL-7Rα were immunoprecipitated. Bound fractions of the γc IPs were analyzed for tyrosine phosphorylation and protein amounts of γc, γc-associated Kit, and γc-associated Jak3 (top panels). Bound fractions of the IL-7Rα IPs were analyzed for tyrosine phosphorylation of IL-7Rα, and IL-7Rα-associated Kit (middle panels). Lysates were analyzed for tyrosine-phosphorylated Kit and the presence of equal amounts of Kit protein at each time point of KL stimulation (bottom panels). (B) KL-induced tyrosine phosphorylation of Jak3 and Stat5. Immunoprecipitations of Jak3 and Stat5 were analyzed for tyrosine phosphorylation and protein amounts of Jak3 (top 2 panels) and Stat5 (bottom 2 panels). (C) Quantification of the relative change in KL-induced protein phosphorylation (γc, IL-7Rα, Jak3, and Stat5) between baseline (0 minutes) and 3 time points of KL stimulation (5, 10, and 15 minutes). Western blot signals on films from 3 independent experiments (■, ♦, ▴) were densitometrically evaluated as described in “Materials and methods, Immunoprecipitations and immunoblotting,” and fold changes were expressed as the log2 ratio. A log2 ratio of 1 is equal to a fold change of 2. The P values for the t tests (5, 10, 15 minutes versus 0 minute, respectively) are indicated by asterisks (*): *.01 ≤ P < .05; **.001 ≤ P < .01; ***P < .001. The connection from time point 0 (fold increase = 1, log2 1 = 0) to time point 5′ is indicated by a dashed line only for orientation as the 0′ value was used for normalization. Thus, mathematically it is not part of the graph anymore. (D) Quantification of the relative change in the amount of phospho-Kit associated with γc and IL-7Rα at 5, 10, and 15 minutes of KL stimulation. Data are displayed and statistically analyzed as in panel C.$

KL stimulation of Jurkat-K7 cells results in the activation of IL-7R, Jak3, and Stat5. (A) γc and IL-7Rα are tyrosine phosphorylated by and associated with Kit after KL stimulation and γc-associated Jak3 is tyrosine phosphorylated in response to KL stimulation. Jurkat-K7 cells were stimulated with KL for the times indicated and γc and IL-7Rα were immunoprecipitated. Bound fractions of the γc IPs were analyzed for tyrosine phosphorylation and protein amounts of γc, γc-associated Kit, and γc-associated Jak3 (top panels). Bound fractions of the IL-7Rα IPs were analyzed for tyrosine phosphorylation of IL-7Rα, and IL-7Rα-associated Kit (middle panels). Lysates were analyzed for tyrosine-phosphorylated Kit and the presence of equal amounts of Kit protein at each time point of KL stimulation (bottom panels). (B) KL-induced tyrosine phosphorylation of Jak3 and Stat5. Immunoprecipitations of Jak3 and Stat5 were analyzed for tyrosine phosphorylation and protein amounts of Jak3 (top 2 panels) and Stat5 (bottom 2 panels). (C) Quantification of the relative change in KL-induced protein phosphorylation (γc, IL-7Rα, Jak3, and Stat5) between baseline (0 minutes) and 3 time points of KL stimulation (5, 10, and 15 minutes). Western blot signals on films from 3 independent experiments (■, ♦, ▴) were densitometrically evaluated as described in “Materials and methods, Immunoprecipitations and immunoblotting,” and fold changes were expressed as the log2 ratio. A log2 ratio of 1 is equal to a fold change of 2. The P values for the t tests (5, 10, 15 minutes versus 0 minute, respectively) are indicated by asterisks (*): *.01 ≤ P < .05; **.001 ≤ P < .01; ***P < .001. The connection from time point 0 (fold increase = 1, log2 1 = 0) to time point 5′ is indicated by a dashed line only for orientation as the 0′ value was used for normalization. Thus, mathematically it is not part of the graph anymore. (D) Quantification of the relative change in the amount of phospho-Kit associated with γc and IL-7Rα at 5, 10, and 15 minutes of KL stimulation. Data are displayed and statistically analyzed as in panel C.

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