FIGURE 8.

Comparison of monomer band intensities between serine and valine replacement mutants. A, the glycosylated monomer bands were evaluated from membrane fractions isolated on sucrose gradients as described in the legend to Fig. 6b. Lane 1, WT; lane 2, C192S; lane 3, C192V; lane 4, C196S; lane 5, C196V; lane 6, C192S/C196S; lane 7, C395S; lane 8, C395V; lane 9, C415S; lane 10, C415V; lane 11, untransfected HEK. B, normalized glycosylated monomer band intensity (gray bars) is plotted in order of decreasing charge density (dotted line with S.E. bars and n values), with the corresponding lane numbers of A shown in parentheses. C192S/C196S, C415S, C196S, C395V, and C192S prestin all show significantly reduced charge density compared with WT (p < 0.05). C, charge density varies linearly with prestin membrane concentration. The glycosylated monomer band intensities from Figs. 6 (B and C) and 8 (A and B) (normalized for equal protein loading) were plotted as a function of mean measured charge density. All values were normalized to the band intensity of WT prestin, and in cases of replicate measures (C395S, C192S/C196S, C415S, C196S, and C192S), the mean band intensity was used. Loss-of-function charge density mutants (C415S, C395V, and C196S) and mutants with abnormally high band intensity (C395S) were omitted (open squares). Linear regression of the remaining functional prestin mutants (closed squares) provided y = 0.0611x + 0.091 (R² = 0.68).