The ERK and PI3K signaling pathways do not control nuclear-cytoplasmic trafficking of NTF2.

<p>(A) HeLa cells were incubated in serum-free DMEM for 4 h (lane 1) and then maintained in serum-free DMEM alone for 4 h, serum-free DMEM containing 150 µM Tween-20 for 4 h (lane 2), or in serum-free DMEM for 4 h and then stimulated with 10% FBS for 30 min (serum) (lane 3). The cells were washed and lysed in the presence of phosphatase inhibitors. 40 µg of each lysate was separated by SDS-PAGE followed by Western blot analysis to monitor the levels of phosphorylated ERK1/2 (p-ERK1/2) (top row) or phosphorylated Akt (p-Akt) (middle row). Analysis of the levels of ERK1/2 (bottom row) was performed as an internal loading control. (B, C and D) HeLa cells were incubated in serum-free DMEM (Untreated), serum-free DMEM containing: 150 µM Tween-20 (Tween-20), 25 µM LY294002 or 50 µM PD98059 for 4 h. The cells were washed and processed for immunofluorescence microscopy to monitor the localization of (B) Ran and (C) NTF2, or for FISH to monitor the localization of tRNA^Lys (D). The nuclei were visualized by DAPI staining. Scale bar represents 10 µm.