TTC9A binds to Tm5NM-1. (A) GST-TTC9A binds to Tm5NM-1-(His)6. COS-7 cells were transfected with Tm5NM-1-(His)6 expression vector or control vector and total cell lysates were collected at 48 h post-transfection. 60 μg GST-TTC9A protein was immobilized onto Glutathione Sepharose 4B gel (Amersham Biosciences) and 300 μg total cell lysates were used for Tm5NM-1-(His)6 pull-down. The proteins bound to the beads were eluted with 2 × SDS-PAGE sample buffer and were separated on an SDS-PAGE gel. Tm5NM-1-(His)6 was detected using anti-His antibody (Amersham Biosciences). GST protein expressed by empty pGEX-5X-3 vector was included as a negative control. 15 μg total cell lysates (5% of input) were loaded in the first lane to indicate the position of Tm5NM-1-(His)6 band. (B) Tm5NM-1-(His)6 pull-down by GST-TTC9A is concentration-dependent. GST-pull down assay was carried out with 2 μg or 20 μg GST-TTC9A as bait protein. The amount of Tm5NM-1-(His)6 pulled down was proportional to the amount of bait protein used. (C) TTC9A-flag interacted with Tm5NM-1-(His)6. Expression vectors for TTC9A-flag and Tm5NM-1-(His)6 were co-transfected into COS-7 cells. Co-immunoprecipitation was carried out with anti-flag agarose beads (Sigma-Aldrich) and Tm5NM-1 was detected by anti-His antibody (Amersham Biosciences). Upper panel: Tm5NM-1-(His)6 was expressed at similar level in control vector and TTC9A-flag transfected COS-7 cells; lower panel: Tm5NM-1-(His)6 was pulled down by TTC9A-flag. (D) TTC9A-flag interacted with endogenous Tm5NM-1-(His)6. Expression vector for TTC9A-flag was transfected into COS-7 cells. Co-immunoprecipitation was carried out with anti-Tm5NM-1/2 (Chemicon) antibody and TTC9A was detected by anti-flag antibody (Sigma-Aldrich). Co-immunoprecipitation with goat pre-immune serum was included as a negative control.