Figure 2
From: Retrograde trafficking of β-dystroglycan from the plasma membrane to the nucleus
Nuclear β-DG derives from the PM. (A) C2C12 cells were incubated with biotin for the indicated time intervals to label cell surface proteins (see Methods). Cells were then subjected to subcellular fractionation to isolate nuclear and non-nuclear fractions and biotinylated proteins were pulled-down using streptavidin-agarose beads and analyzed by SDS-PAGE/Western blotting using primary antibodies for non-phosphorylated-β-DG (upper panel) and phosphorylated β-DG (lower panel). Lower panel blot was reorganized so that the time points order matched the ones shown in upper panel, original blot is shown in Supp. Figure 1. Input: immunoblotting analysis of cellular fractions prior to streptavidin-mediated pull-down. B: Bound/precipitated fraction. Membranes were stripped and reprobed for lamin A/C and GAPDH/calnexin as purity controls for nuclear and non-nuclear fractions respectively. (B) C2C12 cells cultured on glass coverslips were double-immunostained for total β-DG and the early endosomal marker EEA1. Nuclei were counterstained with DAPI prior to CLSM analysis. A typical single Z-section from three independent experiments is shown. Scale bar 20 µm.
