Figure 4.

Pcdh-15+cdh-23 complex formation, its Ca²⁺-dependence, and the role of the deafness mutation R113G, probed using analytical size exclusion chromatography (SEC). Individual traces represent independent experiments. a, top, SEC traces for pcdh-15 and cdh-23 with Ca²⁺ (red and blue) or with 5 mM EGTA (purple and cyan). A shift upon Ca²⁺ removal by EGTA was observed for pcdh-15 (purple vs. red curves). middle, SEC traces for pcdh-15+cdh-23 in the presence of Ca²⁺ (light green) or 5 mM EGTA (dark green). The summation of a purple and a cyan curve from above is shown as a dashed line. The EGTA-treated complex behaved as the sum of its EGTA-treated components, indicating Ca²⁺-dependent complex formation. bottom, Coomassie-stained SDS-PAGE of eluted fractions from EGTA-treated proteins. b, top, SEC traces for mutant pcdh-15_R113G alone (maroon), and mixed with cdh-23 (orange). Wild-type proteins from (a) are shown for comparison. bottom, Coomassie-stained SDS-PAGE of eluted fractions aligned to chromatogram. A reproducible shift in elution volume was observed for the wild-type (green) but not for the mutant mixture (pcdh-15_R113G+cdh-23; orange). The shifted peak (1.61 ml) contained both proteins (1.56 to 1.64 ml fractions).