IMPβ1 recognition by Ei24 is inhibited in the presence of GTPγS or recombinant RanGTP. (A) Lysates from HeLa-Bcl_XL cells expressing GFP, GFP-Ei24, or GFP-IMPβ1 fusion proteins prepared 20 h after transfection were subjected to IP with GFP-Trap resin in the absence or presence of 1.7 mM GTPγS. Western analysis was performed on input and immunoprecipitates (IP: GFP) using the specific antibodies indicated. (B) Densitometric analysis was performed on images such as those shown in A for binding of endogenous IMPβ1 (left) and IMPα2 (right) to GFP-Ei24 or GFP-IMPβ1, as indicated. Pooled results (n ≥ 2) representing the mean ± SD (error bars) for IMP bound relative to no GTPγS treatment (No add.) are shown; p-values (Student’s t test) denote significant differences. NS, not significant. (C) Lysates from HeLa-Bcl_XL cells expressing GFP-Ei24 or GFP alone were incubated for 20 min with 3 µM recombinant of Ran loaded with GTPγS or GDP, before IP and Western blot analysis as in A.