B & N analysis confirms the minimal lateral mobility of Slc26a5-YFP in <i>Slc26a5</i>^<i>YFP/+</i><i>(+neo)</i> mice.

<p>(A) YFP fluorescence distribution in a temporal bone preparation from a <i>Slc26a5</i>^<i>YFP/+</i><i>(+neo)</i> mouse at P38 is shown. (B) Variance of the image series (225 frames) divided with the average brightness (σ²/μ) is shown corresponding to A. (C) YFP fluorescence distribution in Min6m9 cells expressing cytosolic YFP (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005500#sec013" target="_blank">Materials and Methods</a>) is shown. The image was taken under an identical condition with (A). B & N analysis for cytosolic YFP in Min6m9 cells (C) is shown in (D). Scale bars express 20 μm.</p

Ultra-structural analysis of the OHC lateral wall in isolated OHCs.

<p>TEM studies in untreated (A-E), salicylate-treated (F-J), MβCD-treated (K-O) salicylate/MβCD-treated (P-T) isolated OHCs from the mice at P22-23 are shown. Black squares in A, F, K, P were enlarged in B, G, L, and Q, respectively. Asterisks indicate mitochondria. Arrows indicate assumptive pillars. Scale bar expresses 2 μm in A, F, K, and P and 200 nm in B-E, G-J, L-O and Q-T.</p

Slc26a5-YFP is functional in Slc26a5-YFP mice <i>(+neo)</i>.

<p>(A-C) NLC using isolated OHCs from <i>Slc26a5</i>^<i>YFP/+</i><i>(+neo)</i> mice at P20-P26. (A) NLC in isolated OHCs from <i>Slc26a5</i>^<i>+/+</i> (black dotted lines), <i>Slc26a5</i>^<i>YFP/+</i><i>(+neo)</i> (red dotted lines), and <i>Slc26a5</i>^{<i>YFP/YFP</i>}<i>(+neo)</i> (green dotted lines) cochleae is shown. Smooth lines were obtained by fitting to a second order Boltzmann function where population Q_max and α are shown in (B) and (C) respectively. Black (<i>Slc26a5</i>^<i>+/+</i>), red (<i>Slc26a5</i>^<i>YFP/+</i><i>(+neo)</i>), and green (<i>Slc26a5</i>^{<i>YFP/YFP</i>}<i>(+neo)</i>) bars express the mean (± S.E.M.). Values are the mean ± S.E.M; (D) ABR thresholds of wildtype and Slc26a5-YFP <i>(+neo)</i> mice at P18 are similar (E) ABR thresholds of wildtype and Slc26a5-YFP <i>(-neo)</i> mice at P18 are similar. Values are the mean ± S.E.M.; ***: P<0.001, *: P<0.05 by two-way ANOVA followed by student's t test with a Bonferroni correction.</p

Slc26a5-YFP recapitulates endogenous Slc26a5 distribution and is functional in <i>Slc26a5</i>^<i>YFP/+</i><i>(+neo)</i> mice.

<p>(A) Schematic illustration shows morphology of OHCs and the OHC lateral wall. The lateral wall of OHCs consists of a unique trilaminate structure composed of PM, actin-spectrin cortical lattice (CL), and SSC. A more detailed model is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005500#pgen.1005500.s009" target="_blank">S9 Fig</a>. (B-D) Slc26a5-YFP fluorescence (green) in the apical turn of the cochleae from <i>Slc26a5</i>^<i>YFP/+</i><i>(+neo)</i> mice at P20. White square in B is enlarged in C. The dashed line in C indicates the position of the optical xz-plane shown in the inset. Myo7a (red) is labeled as a HC marker in D. Counter-staining of nuclei (blue) using DAPI is shown in D. The dashed line in D indicates the position of the optical xz-plane shown in the inset.</p

Slc26a5 mobility analysis in the lateral wall of isolated OHCs at P18-22.

<p>FRAP examples for MβCD-treated (A), latruculin A-treated (B), diamide-treated (C), latrunculin A/ diamide-treated (D), salicylate-treated (E), MβCD/salicylate-treated (F), diamide/latrunculin A/MβCD-treated (G), diamide/latrunculin A/salicylate-treated (H) OHCs from <i>Slc26a5</i>^<i>YFP/+</i><i>(+neo)</i> mice are shown. Scale bar expresses 10 μm. The dashed line in (F) indicates the position of optical section shown in insets. (I) The normalized fluorescence recovery curves for images A-H are shown (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005500#sec013" target="_blank">Materials and Methods</a>). White arrows in A-H show bleached spots and the black arrow in I indicates the time of bleaching. Error bars express S.E.M. OHC length (J) and Slc26a5 expressing area (K) after each treatment is shown. Values are the mean ± SEM.</p

Topology, molecular weight, and mobile fractions before and after treatment of Slc26a5 and four heterologously expressed membrane proteins.

<p>Mobile fraction for each molecule was shown either before or after co-treatment of MβCD and salicylate. Molecular weight (M. W. in kDa) was predicted based on amino acid sequences. Means and S.E.M. are expressed.</p

FRAP analysis reveals that Slc26a5 has little lateral mobility in the isolated <i>Slc26a5</i>^<i>YFP/+</i><i>(+neo)</i> OHCs.

<p>FRAP examples using the mice at P18-22 for untreated (A) and PFA-treated (B) OHCs are shown. (C) The normalized fluorescence recovery curves for Slc26a5-YFP based on analysis of the bleached spots (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005500#sec013" target="_blank">Materials and Methods</a>). White arrows (A-B) show bleached spots and the black arrow (C) indicates the time of bleaching. Error bars express S.E.M. Scale bar expresses 10 μm. Numbers (n) of OHCs used in two mice from two litters were shown.</p