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. 2010 Feb 8;188(3):325–333. doi: 10.1083/jcb.200910083

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© 2010 Tsai et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 4. — EGF activates nuclear SHP-2 to mediate complex formation of Grb7–HuR–CRM1. (A) Western blot analysis detecting nuclear or cytoplasmic Grb7 as well as tyrosine phosphorylated Grb7 in P19 cells treated with (top) or without (bottom) LMB and EGF as indicated. KOR was monitored, and α-tubulin and lamin B provided the fractionation controls. The RT-qPCR result of KOR mRNA from nuclear (top right) or cytoplasmic (bottom right) fractions of P19 cells treated with EGF is shown on the right (*, P < 0.05). (B) Phosphatase assay from immunoprecipitated nuclear and cytoplasmic SHP-2 from control or EGF-treated P19 cells (*, P < 0.05). The precipitation efficiency and input control are shown on the bottom. (C) Western blot analysis detecting phospho-tyrosine and Flag from the nuclear fractions of P19 cells transfected as indicated with or without EGF treatment. α-Tubulin and lamin B served as the fractionation controls. (D) Autoradiography of in vitro phosphatase assay on in vitro–prepared, anti-Flag antibody–precipitated WT or DM Flag-Grb7 in the presence or absence of SHP-2 or -1 and the SHP inhibitor NSC-87877. A similar amount of input protein was confirmed by Western blotting with an anti-Flag antibody and is shown on the bottom. The predicted signals of Flag-Grb7 are indicated by an arrow, and the nonspecific signal is marked by pound sign. (E) Western blot analysis of Grb7 or CRM1 immunoprecipitation (IP) from nuclear extracts of P19 cells transfected as indicated. A one-eighth input is shown on the bottom. (F) Western blots analysis of nuclear and cytoplasmic fractions (top) and RT-qPCR assay of KOR (bottom left) or actin (bottom right) mRNA from P19 cells transfected as indicated with or without EGF treatment. (A, B, and F) Error bars represent SDs. CY, cytoplasmic fraction; IB, immunoblot; NE, nuclear fraction.