Fig. S4
Fast muscle-specific expression of human mysterin restores the fast muscle malformation and motor deficits of mysterin morphants. (a) RT-PCR analysis of the knockdown efficiency (2.1 ng of MO) in morphants. The lower bands represent the impairment of splicing. (b) The expression level of human mysterin in fast muscle was examined by Western blotting using an anti-FLAG antibody. Protein extracts of Mst-3×FLAG-expressing HEK293 cells1 was loaded for an indication of mysterin products (591kDa; arrow). Mysterin-derived fragments which are not observed in control lanes are indicated by stars. GAPDH is the loading control. (c) Immunostaining of Mst-3×FLAG that is specifically expressed in fast muscle. Control and human mysterin-expressing morphants are stained using an anti-FLAG antibody (white arrows). A single layer and superimposed 3D image are shown separately. RFP is a marker of GAL4 activity. Human mysterin-3×FLAG is distributed in the cytosol, which is consistent with our previous observation in human embryonic kidney 293 cells1. (d) EM of vertical sections of human mysterin-expressing morphants. Fast muscle-specific expression of human mysterin-3×FLAG rescues malformation of muscle fibers. Lower panels are magnified images. (e) Swimming motility (mm/s) of human mysterin-expressing morphants. |