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. 2003 Oct 20;198(8):1189–1200. doi: 10.1084/jem.20030471

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Copyright © 2003, The Rockefeller University Press

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Figure 2. — The Y500F mutation selectively impairs activation of the IRS-2/AKT signaling cascade. (A–D) IL-4–induced signaling events in WT and Y500F T cells. (A) Jak1 and IL-4Rα tyrosine phosphorylation. (Top) Lysates of T cell lymphoblasts were directly blotted with an anti–phospho-Jak1 antibody (pJak1) then reprobed with an anti-Jak1 protein antibody. (Bottom) IL-4Rα immunoprecipitates were probed with an antiphosphotyrosine antibody (pY-IL-4Rα) then reprobed with an anti–IL-4Rα antibody. The IL-4Rα Y500 lysates were overloaded to reveal low level phosphorylation of mutant IL-4Rα in response to IL-4 treatment. (B) IRS-2 and Dok-R tyrosine phosphorylation. IRS-2 and Dok-R immunoprecipitates were probed with an antiphosphotyrosine antibody followed by anti–IRS-2 and anti–Dok-R antibodies, respectively. (C) Akt phosphorylation. Cell lysates were directly blotted with an anti–phospho-Akt serine 473 antibody (pAkt) and then reprobed with an anti-Akt protein antibody. (D) Time course of Stat6 phosphorylation. Stat6 immunoprecipitates were probed with an anti–phospho-Stat6 antibody (pY-Stat6) and then reprobed with an anti-Stat6 antibody. (E–H) IL-4–induced signaling events in WT and Y500F splenic B cells. (E) Tyrosine phosphorylation of IRS-2 (left) and Dok1 (right). (F) p46Shc phosphorylation. Lysates were probed with anti–phospho-Shc antibody and then reprobed with an anti-Shc antibody. (G) Akt phosphorylation on serine 473. (H) Time course of Stat6 phosphorylation.

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