Nuclear lysates were prepared as for co-IP/mass spectrometry analysis except that lysates were precleared with 5 μg of mouse IgG1 isotype control (02-6100, Thermo Fisher Scientific), and precipitations were performed with 4 μg of anti-DUX4 C-2 antibody or 4 μg of isotype control. Western blots were performed by separating samples on 12% Bolt Bis-Tris Plus precast gels (no. NW00122BOX, Thermo Fisher Scientific) in Mops buffer (NP0001-02, Invitrogen), transferring to polyvinylidene difluoride membranes, and probing according to standard methods. Blots were probed with 1:1000 dilutions of anti-C1QBP (A302-862A, Bethyl Laboratories) or anti-DUX4 clone E5-5 (ab124699, Abcam) antibodies, and enhanced chemiluminesence was performed using a mouse anti-rabbit–horseradish peroxidase secondary antibody (31464, Thermo Fisher Scientific), a SuperSignal West Femto kit (34094, Thermo Fisher Scientific) and a ChemiDoc MP Imaging system.

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