Nuclear translocation of endogenous β-DG induced by active ezrin is dependent on IMPβ1.

<p>C2C12 myoblasts stably transfected with vector expressing either the control or importin β1 (IMPβ1) RNAi were cultured on glass coverslips, fixed and immunostained for IMPβ1 <b>A</b> or β-DG <b>B</b>, using FITC-conjugated secondary antibody (green), with nuclei stained using DAPI (blue). Cells were imaged by CLSM, with typical single Z-sections shown (scale bar is 10 µm). <b>B.</b> Quantitative analysis to determine the nuclear to cytoplasmic ratio (Fn/c) of β-DG was performed in control- and RNAi IMPβ1-transfected cells (bottom panel), as per the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090629#pone-0090629-g002" target="_blank">Figure 2</a>. Results represent the mean +/– SD (n &gt; 50 cells) from a series of three separate experiments, with significant differences between cells expressing the control or IMPβ1 RNAi determined by Student t-test. <b>C.</b> Cytoplasmic and nuclear fractions obtained from cells stably expressing either the control or IMPβ1 RNAi and transiently expressing GFP or Ez-T567D-GFP fusion proteins were analyzed by SDS-PAGE/Western using an anti-β-DG antibody (upper panels). Membranes were stripped and reprobed with antibodies against calnexin (Clnx) and Sp3, loading controls for cytoplasmic and nuclear lysates respectively. Nuclear to cytoplasmic ratio (n/c) of β-DG levels were quantified and plotted (bottom panel), as per the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090629#pone-0090629-g002" target="_blank">Figure 2</a>. Results represent the mean +/– SD from a series of three separate experiments, with significant differences between cells expressing the control or IMPβ1 RNAi determined by Student t-test.