Nuclear export of Grb7 is mediated by CRM1 and HuR. (A) Western blot analysis of coimmunoprecipitation of Flag-tagged CRM1–, transportin 1–, or transportin 2–transfected P19 cell lysates with anti-Grb7 or anti-HuR antibody. (B) Western blot analysis of coimmunoprecipitation of Grb7 and HuR from P19 cells with an anti-CRM1 antibody. (C) Immunohistochemistry of DRG neurons (top) and P19 cells (bottom) treated with or without LMB. White dotted lines outline the cytoplasm of individual cells. Colocalization of Grb7 and DAPI was quantified with Pearson correlation coefficients and is shown on the right (*, P < 0.05). Error bars represent SDs. (D) Western blot analysis of the nuclear and cytoplasmic fractions of P19 cells from cultures treated with or without LMB. (E) Western blot analysis of the nuclear and cytoplasmic fractions of P19 cells treated with EGF or EGF plus LMB. (D and E) α-Tubulin and lamin B served as the fractionation controls. (F) Western blot analysis of CRM1 immunoprecipitation from P19 cells transfected as indicated. (G) Western blot analysis of control or HuR-silenced P19 cell lysates pulled down by GST or GST-CRM1. The GST protein input is shown on the right. Predicted GST proteins are marked with arrows. (H) Western blot analysis of anti-CRM1 precipitated from P19 cells transfected as indicated. (A, B, and F–H) A one-eighth input control is shown on the bottom. Ctrl, control; IB, immunoblot; IP, immunoprecipitation. Bars, 25 µm.