FIGURE 4.

EHD4 directly binds to CDH23 in a Ca²⁺-sensitive fashion, and this binding is dependent on the EH domain of EHD4. HEK293T cell lysates from Myc-EHD4-expressing (EHD4) and Myc-EHD4+FLAG-CDH23-expressing (EHD4+CDH23)/Myc-EHD4ΔEH-expressing (EHD4ΔEH) and Myc-EHD4ΔEH+FLAG-CDH23-expressing cells were subjected to co-immunoprecipitation using 2 μg of anti-FLAG antibody and protein A-Sepharose (beads) by rotating at 4 °C for overnight. A, co-IP of Myc-EHD4 and CDH23 where EHD4 was visualized using a goat anti-EHD4 antibody. Lanes 1 and 2 show proteins from the flow-through fraction. Lanes 3 and 4 are eluates from CDH23-anti-FLAG-beads. EHD4 protein was detected by anti-EHD4 antibody and co-immunoprecipitated with CDH23 only in the no Ca²⁺ condition (1 mm EDTA), i.e. not in 1 mm Ca²⁺. The lysate containing only overexpressed Myc-EHD4 was used as a negative control. B, co-IP of Myc-EHD4ΔEH and CDH23 shows no EHD4ΔEH protein in the eluates (lane 3) from CDH23-anti-FLAG-beads when blotted with anti-Myc-HRP-conjugated antibody. The lysate containing only overexpressed Myc-EHD4ΔEH was used as a negative control (lane 4). Lanes 1 and 2 show proteins from the flow-through fraction. EHD4ΔEH protein was not co-immunoprecipitated either in the presence of 1 mm EDTA or 1 mm CaCl₂ demonstrating that the EH domain of EHD4 is required for the Ca²⁺-sensitive CDH23-EHD4 binding.