GIFT15 and receptor-mediated signaling. (A) BIAcore analysis of IL-15Rα chain interaction with rIL-15 and purified GIFT15. Representative sensorgrams for the binding of (A) 12, 25, or 50 nM rIL-15 or (B) 15, 31, or 46 nM purified GIFT15 to 1300 RU rIL-15Rα–Fc as detected by SPR. (B) GIFT15-induced phosphorylation of STAT3/5 protein. Splenocytes (10⁶) were stimulated for 15 minutes with 30 pmol rIL-15, rGMCSF, or both cytokines inoculated in B16-GFP CM or with B16-GIFT15 CM, and cell lysate was probed for phosphorylated STAT3/STAT5. Total STAT3 or STAT5 protein was used as loading control. GIFT15-induced phosphorylation of STAT5 protein. (C) GMCSFR-mediated signaling. JAWS-II cells (10⁶) were stimulated as performed previously with primary splenocytes, and cell lysate was probed for phosphorylated STAT5. Total STAT5 protein was used as loading control. (D) Structural model of GIFT15 (green, gray, and cyan ribbon) complexed with IL-15Rα (yellow ribbon), IL-2β (purple ribbon), and IL-2Rγ (red ribbon).