Fig. 2. Tissue expression and isoforms of myotubularin. (A) Three mg of total protein extracts from different tissues of a 14-week-old mouse were immunoprecipitated with 1G1 antibody and loaded on a 8% SDS-PAGE gel. Myotubularin was detected after western blotting with the 2D2 antibody (1/2000). Below, a longer migration reveals the presence of a doublet specific to muscle (and heart, not shown). Different percentages of acrylamide and gradients tested did not resolve the two bands any better. The arrow indicates the migration of the 603 amino acid myotubularin construct transfected into cells, representing the common isoform. (B) The muscle-specific isoform appears after myotube formation. C2C12 mouse myoblasts were differentiated at confluence in DMEM+5% FCS into myotubes, and myotubes were stimulated with insulin like growth factor (IGF1 from Calbiochem at 25 ng/ml) as indicated. Protein extracts were prepared at different time points during differentiation and compared with adult muscle and liver tissues.