Fig. 3. Myotubularin is cytoplasmic and associated with plasma membrane. (A) Wild-type untagged myotubularin overexpressed in COS cells was detected with the specific 1G6 monoclonal antibody (1/1000) followed by Cy3-conjugated goat anti-mouse antibody (1/300). Confocal microscopy analysis does not show any nuclear signal. The inactive C375S mutant was also localized as a dense cytoplasmic network in different cell types (COS, HeLa, 3T3 fibroblasts, myoblasts and myotubes). Also note the labeling of plasma membrane. Preimmune serum or immunizing peptide competition (100 µg, 30 minutes, room temperature) abolished the signal. (B,C) Localization in transfected C2C12 mouse myoblasts and myotubes respectively. (D) Transfected COS cell showing altered cell shape and presence of myotubularin in extended filopodia. This pattern was also observed with inactive myotubularin mutants.

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