FIGURE 2.

Cysteine replacement mutants membrane localize. GFP, di-8 (membrane), and bright field images (columns from left to right) are shown for WT and C192S prestin-expressing HEK cells (first and last row, respectively). In both cases, the fluorescence of the prestin-GFP fusion protein coincides with that of di-8, a lipid-like probe that partitions into the cell membrane. This demonstrates successful trafficking of the protein, which is clearly evident when thresholding is performed to remove low intensity pixels from each fluorescent channel (the lowest ∼20%), and the remaining colocalized signal is displayed (fourth column). Native HEK cells labeled with di-8 (second row) and unlabeled HEK cells expressing prestin (third row) are shown to demonstrate efficient spectral separation of GFP and di-8 fluorescence into their respective channels. Despite reduced NLC for C192S prestin, the extent of membrane localization is visually indistinguishable from WT prestin. The membrane localization of C192S prestin is representative of the trafficking observed for each of our mutant prestin constructs (see supplemental Fig. S1). Scale bars, 20 μm.