(a) Coomassie-stained SDS–PAGE gels showing purification of His-drp6 (WT) and His-drp6-DTD (DTD) expressed in Escherichia coli. M is the molecular weight markers. Some of the markers are indicated on the sides. The purification of His-drp6-DTD was partial and contained additional proteins from E. coli, including one prominent band below 28 kDa. The purified His-drp6-DTD appearing below 17 kDa marker is indicated by an asterisk. (b) Lipid overlay assay as detected by western blot analysis using anti-his antibody. (Tt-Lip); total Tetrahymena lipid spotted on nitrocellulose membrane and incubated with His-drp6 in absence (top) or presence (middle) of GTP. The bottom spot is incubated with BSA. Strip spotted with 15 different lipids and incubated either with His-drp6 (Drp6) or with His-drp6-DTD (DTD). Both Drp6 and DTD interacted with PA, PS, and CL are indicated. (c) Floatation assay using liposomes containing 70% phosphatidylcholine and 20% phosphatidylethanolamine additionally supplemented with 10% CL (PC/PE+CL), 10% PA (PC/PE+PA), or 10% PS (PC/PE+PS). While liposomes in (PC/PE) contained 80% phosphatidylcholine and 20% phosphatidylethanolamine, no liposome was added in (–Liposome). His-drp6 was incubated either with different liposomes or without liposomes, overlaid with sucrose gradient, and subjected to ultra-centrifugation. Fractions were collected from top and detected by western blot analysis using anti-his antibody. Drp6 appearing in the top four fractions indicate interaction with liposome. The experiments were repeated at least three times, and representative results are shown here. (d) Lysates of Tetrahymena cells expressing either GFP-drp6 (top) or GFP-drp6-DTD (bottom) were fractionated into soluble (S) and membrane (P) fractions and detected by western blot using anti-GFP antibody. Molecular weights of the proteins are indicated on the right.