FIGURE 6.

IL-2Rβ Thr-450 phosphorylation is a positive regulator for activation of downstream signaling molecules, receptor complex formation, and STAT5B transcriptional activity in a reconstituted HEK293 cell system. HEK293 cells were transfected with γc, JAK3, STAT5B, and IL-2Rβ (WT or T450A) and incubated for 48 h in complete medium, followed by 48 h of incubation in 1% FBS medium. Transfected cells were stimulated without (−) or with IL-2 (+) for 10 min. A, whole cell lysates were separated by SDS-PAGE and Western-blotted as indicated. B, IL-2Rβ was immunoprecipitated (IP), separated by SDS-PAGE, and analyzed by WB for coimmunoprecipitation of JAK3, STAT5, and γc as indicated. Input controls for immunoprecipitated JAK3 and STAT5 are indicated. Representative data from three independent experiments are shown. C, HEK293 cells were transfected with IL-2Rβ (WT or T450A), γc, JAK3, STAT5B, β-casein-luciferase, and pCMV-β-galactosidase. 48 h post-transfection, cells were incubated with or without IL-2 for an additional 48 h, and luciferase activity was measured and normalized to β-gal activity. Each treatment was performed in triplicate. Results are presented as the mean ± S.D. of three independent experiments. Statistical significance was determined using Student's t test. *, p < 0.05.