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Figure 3


Fig. 3. Immunostaining of WT and heterozygous targeted cells shows myofibrillogenesis is normal. WT and heterozygous targeted cells (clone 5-A8) were coimmunostained for {alpha}-actinin and skeletal myosin or {alpha}-actinin and M-band titin, and for actin using Alexa-546 phalloidin, or stained for titin (Z1-Z2 antibody) myomesin and myosin, and imaged using a deconvolution microscope. Arrows show the positions of the Z-disks in each of the images. In the top four panels, the Z-disks stain positively for {alpha}-actinin and actin (thus, purple in the merged image). Myosin staining is clearly defined by a doublet between the Z-disks stained for {alpha}-actinin. The M-band region of titin is clearly seen as a stripe at the middle of the sarcomere between the two Z-disks stained for {alpha}-actinin. In the bottom two panels, the Z-disks stain immunopositively for titin (Z1-Z2 antibody), and myomesin is found midway between the Z-disks at the M-band. Occasional weak staining with the Z1-Z2 antibody at the M-band, which we assume is an anomalous cross-reaction, is sometimes observed. Bars, 5 µm.





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