Inhibition of IL-3–stimulated tyrosine phosphorylation of Shc by different Shc mutant forms.

(A) Schematic representation of Shc mutations expressed as FLAG-tagged proteins. (B) IC2 cell lysates from each FLAG-tagged Shc infection were subjected to Western blotting with anti-FLAG antibodies to confirm expression of the different FLAG-tagged Shc proteins. (C) Total cell lysates from cells expressing different Shc mutant forms were unstimulated (−) or stimulated (+) with IL-3 for 5 minutes and analyzed by immunoblotting with anti-pTyr (upper panel) or anti-FLAG antibodies (lower panel). WT indicates wild type; 2M, R175/401M; 2F, Y239/240F; and 3F, Y239/240/317F.