Figure 4.

Tim-3 cytoplasmic tail tyrosines are required to augment NF-κB, NF-AT/AP1, and IL-6 transcriptional activation in a Syk-dependent manner. (A) MC/9 mouse mast cells were transfected with empty vector (pCDEF3) or one of the indicated Flag-tagged Tim-3 constructs. Transfected MC/9 mast cells were stimulated with IgE and 50 or 100 ng/ml of DNP₃₂-HSA, with or without addition of anti-Flag Ab for 6 h. (B and C) MC/9 mast cells were cotransfected with luciferase reporters for NF-κB (B) or NFAT/AP-1 (C). (D–G) MC/9 mast cells were cotransfected with empty vector (pCDEF3) or full-length Flag-tagged Tim-3 and an IL-6 promoter luciferase reporter. Transfected cells were stimulated with IgE and Ag in the presence or vehicle control (DMSO), BAY61-3606 (E), or PP2 compounds (F) for 6 h. (G) MC/9 mast cells were transfected with the indicated Tim-3 constructs and IL-6 promoter luciferase reporter and stimulated as described above. Results are representative of three independent experiments performed in triplicate. **, P < 0.005; ***, P < 0.0005.