*2.3. Subcellular Localization of the Single Amino Acid rAbcc6 Mutants That Showed Reduced ATP Efflux Activity*

To exert its function, ABCC6 needs to reside in the plasma membrane. Confocal microscopy demonstrated that all rAbcc6 mutants with reduced activity routed to the plasma membrane, similar to wild-type rAbcc6 (Figure 6). This indicates that reduced plasma membrane localization was not the underlying cause of the reduced activity of these rAbcc6 mutants. Notably, although a significant proportion of the rAbcc6 mutant proteins also resided in intracellular compartments, this was not different from wild-type rAbcc6 and is consistent with our previous observations [19].

**Figure 6.** Subcellular localization of rAbcc6 mutants with reduced ATP efflux activity. Representative images of the

subcellular localization of wild-type and single mutant rAbcc6 in HEK293 cells, as determined by confocal microscopy using the K14 anti-rAbcc6 rabbit polyclonal antibody. Red: Na+/K<sup>+</sup> -ATPase, a marker for the plasma membrane; Green: rAbcc6.; Blue: DAPI nuclear staining; wt rAbcc6: wild-type rAbcc6, control: parental HEK293 cells. All scale bars represent 30 µm.
