Fig. 4. Subcellular distribution of endogenous myotubularin in (A) lymphoblasts from normal (L1421) or an XLMTM patient deleted for exons 1-13 of the hMTM1 gene (89-441), (B) HeLa cells and (C) mouse myotubes. Subcellular fractions were prepared as described in Materials and Methods and enrichment was confirmed under the microscope (example below the western blot in A) and with protein markers (tubulin is present in the same fraction as myotubularin). Myotubularin was immunoprecipitated from the different fractions with 1G1 antibody and detected on a western blot by 1G6 (1/10,000) for the human cell lines and 2D2 (1/2000) for the mouse myotubes. P1, nucleus; P2, big organelles; S1, cytoplasm and all organelles; S2, cytoplasm and small organelles; T, total extract; TR, myotubularin overexpressed in COS cells. (D) Cytoskeleton fractionation of HeLa cells transfected with wild-type (WT) and substrate-trap mutant (D278A) myotubularin. Actin-based microfilaments and intermediate filaments containing vimentin were recovered in the cytoskeletal (P) fraction, whereas actin monomers and tubulin were recovered in the cytosolic (S) fraction. (E) The same cytoskeleton fractionation applied to mouse C2C12 myotubes. Myotubularin was immunoprecipitated and detected as above.