Cell culture plates and slides were coated with the cardiac extracellular matrices (20 μg/ml, final concentration) described above. Myocyte cultures were treated with or without IRF5D (50 μg/mL, 24 h). IRF5 translocation from the cytosol to the nucleus was determined on cultured myocytes using immunofluorescence confocal microscopy. Anti-IRF5 antibody (Santa Cruz Biotechnologies) was used as the primary antibody and Alexa 488-labeled goat anti-mouse IgG antibody (Molecular Probes) was used as the secondary antibody and DAPI as a nuclear stain. Fluorescent images were captured with a fluorescent Nikon Eclipse microscope and fluorescence intensity in the cytosol and nuclei of the cells in the captured images was measured using NIKON Element imaging software as previously described [12 (link)].
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