Immunoblotting of DUX4 and DUX4c Proteins

Twenty micrograms of total protein extracts were separated by electrophoresis (4–12% PAGE-SDS) in MOPS buffer at 100 V for 3h30 and transferred to a nitrocellulose membrane at 260 mA for 1h45 in a blotting buffer (PBS, 25 mM Tris, 192 mM Glycine, 20% methanol). Protein transfer was confirmed by Ponceau red staining of the membrane. After rinsing in PBS and blocking with PBS-milk 5% for 1 h at RT, the membrane was incubated overnight with either primary antibodies: MAb 9A12 mouse anti-DUX4 (1/1000), rat anti-DUX4c 860 serum (1/1000), or rabbit anti-DUX4c serum (1/1000) diluted in PBS-2% BSA followed by rinsing in PBS and incubation 1 h at RT with secondary antibodies coupled to HRP at 1/5000 dilution. Revelation was performed with either the Super Signal West Femto Maximum Sensitivity Substrate (Thermo Scientific) for endogenous protein detection or Lumi-Light Western Blotting Substrate (Roche) for overexpressed protein detection on Hyperfilm ECL (Amersham).