The chimera was also visualized with an antibody detecting an amino terminus epitope between amino acids 3 and 23 of H,K- ATPase. The researchers harnessed the inability of amino acids 1 through 85 of H,K- ATPase to confer apical distribution by tagging all subsequent chimeras with this sequence. This addition permitted visualization specifically of the chimera yet presumably did not affect localization. Figure 2 depicts localization results for four more chimeras. Chimera IV displays basolateral distribution while chimera V shows apical distribution as seen by immunofluoresence results achieved in the same manner as figure 1.
V constitutes the second ectodomain and the fourth transmembrane domain of the alpha subunit.
Chimeras VI and VII further define this region defined by chimera V. Chimera VI contains only the second ectodomain and localizes basolaterally. As it had been previously demonstrated that complexation with the beta subunit is necessary for ER export, the investigators were interested in establishing the identity of the beta subunit which colocalized with their chimeras. Chimera IV, as expected, exhibited an apical distribution. The beta subunit was present at both the basolateral and apical membrane domains for the transfected cell line. This result supported the investigators prediction that the endogenous beta subunit would colocalize with chimera IV since it contained the carboxyl terminus half of Na,K- ATPase which had been shown to specify interaction with the beta subunit.