Ab124699

In situ proximity ligation assay (PLA) is suitable for quantification of protein expression and for characterization of modifications and interactions of proteins [42 (link), 43 (link)]. PLA assay was performed using Duolink^® PLA Fluorescence (Merck, Darmstadt, Hessen, Germany) according to manufacturer's instruction. In brief, the Reh cells expressing DUX4/IGH or mutants were transfected with LEGO‐iG2 vector containing recombination‐activating gene 1/2 (RAG1/2). The cells were harvested and attached to coverslips through centrifugation (700 rpm, 5 min). The antibodies of DUX4/IGH (bs‐12369R, 1:500 dilution, Bioss, Shanghai, China; ab124699, 1:1500 dilution, Abcam) and RAG1/2 (sc377127 and sc517209, 1:500 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were incubated, then staining with the PLA probes (secondary antibody, one PLUS and one MINUS). If the target proteins interacted with each other, the DNA could be amplified and visualized by fluorescently labeled complementary oligonucleotide probes. The number and intensity of the dots were detected by fluorescence microscopy.

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.

After 1, 2, 3, and 4 days of myogenic differentiation, the cells were fixed in 4% paraformaldehyde for 15 min at room temperature, permeabilized in 2% Triton for 10 min, then washed with PBS. Immunostaining was performed overnight at 4°C with a 1:2000 dilution of DUX4 E5.5 rabbit monoclonal antibody (ab124699, Abcam) in PBS, a 1:250 dilution of MF20 antibody against myosin heavy chain (MYH1E (MF20, DSHB). Cells were washed and labeled with fluorescent-conjugated Alexa 488 or Cy3 anti-rabbit or anti-mouse secondary antibodies for 1 h at room temperature, then washed again. The images were acquired on a Dragonfly 500 Confocal Microscope System using an immersion lens under 60× magnification. A fusion index was calculated for each image with CellProfiler software (v2.1.1) using a custom made analysis pipeline. In short, individual nuclei and larger nuclei clusters were segmented based on Hoechst staining and were identified based on shape and size (see Supplementary Fig. S1). Fusion index was defined as the percentage of individual nuclei located in a nuclei cluster of > 2 nuclei in size. A threshold of > 2 nuclei was used to reduce the false positive labeling of multinucleated myotube nuclei due to a possible slight oversegmentation of the individual nuclei.