Western blots

Patient primary myoblasts and primary fibroblasts were lysed in Radio-Immunoprecipitation Assay (RIPA) Buffer (0.1% SDS, 1% NP40, 0.5% sodiumdeoxycholine, 150mM NaCl, 5mM EDTA and 1× Complete protease inhibitor cocktail (Roche, Basel, Switzerland) in 20 mM Tris pH 7.4. The lysate was sonicated using a Bioruptor Pico (Diagenode, Liege, Belgium) and centrifuged for 10 minutes at 13.500 RPM to pellet the cell debris. Protein concentration was determined using Pierce BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA) and equal protein amounts were boiled in 1× Laemmli Sample Buffer (2% SDS, 10% glycerol, 2% β-mercaptoethanol and 0.01% bromophenol blue in 60 mM Tris pH6.8). Samples were loaded on Criterion TGX 4–20% gels (Bio-Rad, Hercules, CA) and transferred to an Immobilon-FL PVDF membrane (EMD Millipore, Billeria, MA). The membrane was subsequently blocked in 4% skim milk in PBS and probed for SMCHD1 (ab176731, Abcam, Cambridge, UK), α-Tubulin (T6199, Sigma Aldrich, St. Louis, MO) or HSP90 (#4874, Cell Signaling Technology, Danvers, MA) in Immunobooster (Takara Bio Inc. Kusatsu, Japan). Membranes were incubated with donkey-α-rabbit IRDye-800 and donkey-α-goat IRDye-680 (Li-cor, Lincoln, NE) followed by visualization of immunocomplexes using an Odyssey Infrared Imaging System (Li-cor, Lincoln, NE). Protein quantification was performed using the accompanying software package.