Figure 4

Proximity ligation assays of a subset of interactions. A/ Graphs showing the ratio of the signal for the PPI divided by the signal for the prey protein alone with respect to the ROI corresponding to membrane (light grey) or cytoplasm (dark grey) for the bait proteins DYSF, APPL1, MYOM2, OPTN and SGCG. Prey proteins are named with their gene symbol. The line corresponding to 20% is indicated for each graph. For technical reasons, in the case of DYSF/SGCG and MYOM2/TTN interactions, the PPI/prey ratio is calculated as the ratio of SGCG and TTN molecules with respect to DYSF and MYOM2 although DYSF and MYOM2 were found as preys of SGCG and TTN baits. For MYOM2 and OPTN, two TTN antibodies were tested with epitopes at the Z-disc and N2A regions, respectively. Among the 44 tested interactions, 34 showed a “PPI signal” with various proportions between membrane and cytoplasm. B/ Representative confocal images of PLA results. Duolink amplifications are visualized by fluorescence (white dots). Upper panels: PLA+/- labeling of single proteins. Left panel = negative control consisting of DYSF with probes corresponding to the PPI for evaluation of background staining. Middle panel: the DYSF-APPL1 interaction signal showed a strong labeling both at the membrane and the cytoplasm. Right panel: the DYSF-DES labeling showed that the signal is mainly located at the membrane. Scale bars= 20 μm.