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. 2010 Nov 3;99(9):2978–2986. doi: 10.1016/j.bpj.2010.09.003

Figure 2.

Figure 2

Incorporation of DM-cTnC into the myofilament lattice. Panel A illustrates a typical example of SDS-PAGE gel analysis using Blue Silver staining to visualize TnC protein. Due to the fluorescent label, DM-TnC migrated slower allowing separation from WT-TnC. Note that exchange with 100% DM-TnC revealed a small (∼5%) amount endogenous TnC remaining in the fiber (consistent with the virtual complete elimination of active force development in 100% DM-TnC exchanged fibers, c.f. Fig. 3). Fig. 2B illustrates the percent of DM-cTnC incorporated into the myofilament (y axis) as a function of the percent DM-cTnC used in the exchange solution (x axis). Error bars represent the mean ±SE. As shown, the percent of DM-cTnC incorporated into the myofilament was linearly related to the amount of DM-cTnC in the exchange solution. Fig. 2C illustrates confocal images of two fibers labeled with fluorescent WT-cTnC or fluorescent DM-cTnC (i and iv) or rhodamine phalloidin (ii and v), as well as the merge images (iii and vi). Note the sarcomeric pattern of the TnC labeling in comparison to the rhodamine phalloidin staining. Merging the images demonstrates that both bacterially expressed proteins localize to the thin filament when they are allowed to incubate with skinned fibers.