FIGURE 2.

Confocal images show CHO cells transfected with prestin-YFP constructs into which the HA epitope was inserted into position 168 (A–F) and position 371 (G–I). The HA tags were detected with a mouse anti-HA epitope and Alexa 647-conjugated antimouse antibody (A, D, and G). (B, E, and H). YFP images. (C, F, and I) Merged images. Cells in A–C and G–I were stained live, and cells in D-F were fixed and permeabilized with detergent. As is evident, the detection of the HA epitope at position 168 required that the cells be permeabilized, whereas that at position 371 was detectable in live cells without permeabilization. These results suggest that position 168 of prestin in these constructs lies on the intracellular surface, whereas position 371 lies on the extracellular surface. (J) Substitution of the two potential N-glycosylation asparagine residues with glutamine residues does not result in a change in molecular weight, indicating that prestin is not glycosylated at these two residues. CHO cells were transfected with constructs of prestin fused to poly-His V5 epitope tag at its C-terminus (lane A) and prestin N163Q + N166Q double mutant fused to a poly-His V5 epitope tag at its C-terminus (lane B). Normal and mutated prestin were purified from lysed CHO cells on a Ni column and separated by PAGE (8%). The gel was blotted on to polyvinylidene difluoride and probed with an anti-V5 antibody/horseradish peroxidase-conjugated secondary antibody followed by enhanced chemiluminescence detection. As is evident, prestin migrates at its predicted molecular weight of 80 kD, as does the N163Q + N166Q double mutant.