(A) HEK293 cells were transfected with the constructs indicated on top of each panel. Immunoprecipitations were carried out with HA antibody, followed by western blotting to detect co–expressed proteins. The upper row shows CoIP result and the lower rows show input protein. Note strong binding activity was detected between Fam65b and RhoC, weak binding was detected with RhoA, while no binding was detected with Rac1 and Cdc42. (B) OHCs were injectoporated at P2 to express Fam65b–GFP and HA–RhoC. Expression of Fam65b-GFP (green) and HA-RhoC (red) was evaluated 2 days later by immunohistochemistry. Stereocilia were visualized by phalloidin staining (blue). Note colocalization of Fam65b-GFP and HA-RhoC at the base of stereocilia. Scale bar: 4 µm. (C, E, G, H) HEK293 cells were transfected with the constructs indicated on top of each panel. Immunoprecipitations were carried out with Myc antibody (C, E, H) or HA antibody (G), followed by western blotting to detect co-expressed proteins. The upper rows show CoIP results and the lower rows show input protein. Note co-expression of HA-RhoC enhances/stabilizes the oligomerization of Fam65b at N termini (C) but not C termini (E). RhoC did not promote oligomerization of a Fam65b mutant (RL changed to AA; diagram in G) devoid of RhoC binding activity (G, H). (D, F, I) Quantification of CoIP results by scanning of similar gels as shown in (C) (E) and (H). The values are derived by quantifying more than 3 independent experiments. (J) Fam65b-deficient OHCs were injectoporated at P2 to express Fam65b-GFP or mutant Fam65b protein without RhoC binding activity (Fam65bRL/AA-GFP). Expression of Fam65b was evaluated 2 days later by staining for GFP (green). Stereociliary morphology of OHCs was visualized by phalloidin staining (red). Note morphological defects of stereocilia were rescued by Fam65b-GFP but not by Fam65bRL/AA-GFP. Scale bar: 6 μm. (K) Quantification of transfected hair cells from (J). White column represents number of rescued OHCs. (L) Model of Fam65b localization pattern in hair cells. In our model, Fam65b forms a circumferential ring near the basal taper domain of stereocilia, while taperin forms a dense core structure. Fam65b forms oligomers via head-to-head and tail-to-tail interactions and RhoC binds to Fam65b and promotes oligomerization.