Inhibition of IL-2–induced Stat5 phosphorylation by JAB/SOCS1/SSI-1.

We transiently transfected 293T⁺ cells with IL-2Rβ, IL-2Rγ, Jak3, Stat5a, and Stat5b in the presence or absence of WT or R105K or R105E SH2-domain mutants of JAB/SOCS1/SSI-1. After 24 hours, cells were split and plated in duplicate. Another 24 hours later, one set was stimulated with 2 nM IL-2 for 15 minutes, and the other set was left untreated. Cells were then lysed and immunoprecipitated with antibodies against Stat5a and Stat5b, and the immunocomplexes were subjected to SDS-PAGE and Western blotting with antiphosphotyrosine (PY20), anti-Stat5, or anti-Myc 9E10 antibodies.