Fig. 3. Complex formation of Mfn1 protein in solubilized conditions. (A) Assay for GTP-dependent homotypic Mfn1 complex formation. (B) PNS, post mitochondrial supernatant (PMS) and mitochondria fractions (mit) were prepared from HeLa cells expressing HA-tagged or FLAG-tagged Mfn1, and solubilized by digitonin. The lysates with HA-tagged or FLAG-tagged Mfn1 were mixed, and incubated with or without GTP, at 0°C or 30°C for 1 hour. The reaction mixtures were subjected to immunoprecipitation using anti-HA antibodies, and analyzed by immunoblotting using anti-FLAG and anti-HA antibodies. The reaction mixtures were also analyzed by immunoblotting using anti-FLAG antibodies (input). (C) Time course of co-immunoprecipitation. The solubilized PNS or mitochondrial fractions with HA-tagged or FLAG-tagged Mfn1 were mixed and incubated with GTP on ice or at 30°C. Co-immunoprecipitated Mfn1-FLAG by anti-HA antibodies was quantified. (D) Solubilized mitochondrial fractions with HA-tagged or FLAG-tagged Mfn1 were mixed and incubated with 1 mM nucleotides as indicated. The reaction mixtures were analyzed as in B. (E) Wild-type Mfn1 (wt) or the mutant K88T with HA or FLAG tags were mixed in the indicated combinations and incubated with GTP. The reaction mixtures were analyzed as in B. (F) The mitochondria (mit), or salt washed mitochondria (wash mit) were solubilized by digitonin. The solubilized (dig+) or intact (dig–) mitochondria were incubated with or without GTP, and analyzed as in B. (G) The mitochondria harboring Mfn1-HA or Mfn1-FLAG were incubated in the digitonin-solubilized (dig+) or unsolubilized (dig–) conditions with 0.4 mM GTP at 30°C for 1 hour (1st GTP). After incubation, 2 mM GTPS or digitonin was added and further incubated at 30°C for the indicated time intervals (2nd GTPS), then subjected to immunoprecipitation and immunoblotting.