Figure 6.

Time-dependent effects of Pi on Mnk1 translocation between cytosol and nucleus. (A) Western blot analysis of phospho-Mnk1 between cytosol and nucleus. Cells were treated with 10 mM Pi for various time points, collected, then, separated into cytosol and nucleic fractions by Nuclear Extract Kit. Twenty-five micrograms of protein was used for Western blot of phospho-Mnk1. GAPDH and Histone H1 were used as controls for cytosol and nucleus, respectively. Upper panels, Western blot analysis; lower panel, densitometric analysis. (B) Fluorescence imaging of the distribution of phospho-Mnk1 between cytosol and nucleus. At different time points, cells were collected, fixed, and assayed by immunostaining for phospho-Mnk1 (green via FITC), and nuclei (blue via DAPI). Two representative time points are shown for better explanation. Scale bars = 10 μm.