Domains involved in TTC9A and Tm5NM-1 interaction. (A) Western blot analysis of the expression TTC9A truncates in the cell lysates using anti-flag antibody from Sigma-Aldrich. All truncations expressed proteins of predicted sizes. The hollow streak in some of the bands indicates over-saturation of the signal. (B and C) Test of Tm5NM-1-(His)6 pull-down by different TTC9A truncations in pXL-Flag vectors. The TTC9 truncation constructs were transfected into COS-7 cells together with Tm5NM-1-(His)6 expression vector. The interaction between flag-TTC9A truncates and Tm5NM-1-(His)6 was analyzed by co-immunoprecipitation with anti-flag agarose beads (Sigma-Aldrich). The upper panels in B and C are Western blotting analysis of Tm5NM-1 expression in the cell lysates using anti-His antibody (Amersham Biosciences), and the lower panels are the Tm5NM-1-(His)6 co-immunoprecipitated with flag-TTC9A and the truncated proteins. (D) The upper panel is the analysis of Tm5NM-1 expression in the cell lysates; the middle panel is the co-immunoprecipitated Tm5NM-1 with flag-TTC9A and the truncated proteins; the lower panel represents the amount of TTC9A and its truncates pulled down by the anti-flag agarose beads (Sigma-Aldrich) in the co-immunoprecipitation assay.