Journal: PLoS ONE
Article Title: Nuclear Import of β-Dystroglycan Is Facilitated by Ezrin-Mediated Cytoskeleton Reorganization
doi: 10.1371/journal.pone.0090629
Figure Lengend Snippet: 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 A or β-DG 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. 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 . Results represent the mean +/– SD (n > 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. C. 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 . 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.
Article Snippet: The PCR product was digested with SalI and EcoRI, cloned into the pGTH vector and confirmed by sequencing. psi-mH1 vector expressing an interfering RNA (RNAi) specific for IMPβ1 (Cat. # MSH027440-mH1), or an irrelevant RNAi (Cat. # CSHCTR001-mH1) and co-expressing the reporter protein mCherry for identification of transfected cells were purchased from GeneCopoeia, Rockville, MD USA.
Techniques: Stable Transfection, Transfection, Plasmid Preparation, Expressing, Cell Culture, Staining, SDS Page, Western Blot
