Plectin 1 links intermediate filaments to costameric sarcolemma through β-synemin, -dystrobrevin and actin

JCS021634 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. In vitro and in vivo interactions between plectin and β-synemin. (A) Crude IF fraction, obtained by extracting muscle light microsomes (LM) with Triton X-100, was separated on SDS gels, from which a protein of ∼160 kDa was extracted electrophoretically. The extracted protein was reloaded onto SDS-PAGE and blotted onto PVDF membranes, followed by overlay of Myc-tagged N-terminal plectin 1 (PleN1) fragments or β-galactosidase (LacZ). Detection with anti-Myc antibody showed that PleN1 bound the protein (later identified as β-synemin), whereas LacZ did not. (B) In immunoprecipitation experiments, Myc-tagged PleN1 fragments in combination with anti-Myc antibody (Myc mAb) coimmunoprecipitated β-synemin (β-Syn), whereas Myc-tagged PleN1 with control IgG and Myc-tagged LacZ with Myc mAb did not. (C) In reciprocal pull-down assay, GST-fused β-synemin in combination with glutathione beads (Gl-beads) pulled down PleN1, but not LacZ. GST alone did not precipitate PleN1. (D) β-Synemin- and plectin-immune complexes were precipitated from lysates of LM by polyclonal anti-β-synemin and anti-plectin antibodies (pAb), respectively. β-Synemin coimmunoprecipitated plectin and vice versa.

• Supplemental Figure S2 -

  Fig. S2. Aberrant IF networks and abnormal cytoplasmic deposits in dystrophin-deficient CXMDJ muscle fibers. When compared with normal muscle fibers (A-C), dystrophin-deficient muscle fibers exhibited more intense β-synemin staining along the sarcolemma (D-F). In addition, some muscle fibers (asterisks in E) contained aberrant IF networks and abnormal cytoplasmic deposits (D). Aberrant IF networks represent irregular and larger mesh structures, as compared with normal IF networks. Note that cytoplasmic deposits were positive for β-synemin (D), but negative for α-dystrobrevin (E). Most of regenerating fibers, positive for vimentin (H) or developmental myosin (K), contained aberrant IF networks and cytoplasmic aggregates (G and J). These aggregates were positively stained using anti-β-synemin and anti-vimentin antibodies (I), but not using anti-developmental myosin antibody (L). Bars: 10 µm