Coupled oligomerization of a mitochondrial Mid51/Fis1 complex. (A and B) Quantification of immunoprecipitated WT Mid51 (A) and coimmunoprecipitated WT Fis1 (B) in a Mid51/Fis1 complex, with and without Mid51, confirming that IP of Mid51 and co-IP of Fis1 is dependent on the presence of Mid51 (lane 2); n = 3 independent experiments. See Fig. S3 A for representative blots. (C and D) Quantification of immunoprecipitated WT Mid51 (C) and coimmunoprecipitated WT Fis1 (D) in a Mid51/Fis1 complex, with and without Fis1, confirming that co-IP of Fis1 is dependent on the presence of Fis1 (lane 2); n = 3 independent experiments. See Fig. S3 B for representative blots. (E) IP of myc-tagged WT Mid51 and co-IP of Flag-tagged Fis1(WT) or mutant Fis1(LA), with corresponding input. *, Nonspecific bands. (F and G) Protein quantification and stoichiometry of the immunoprecipitated mitochondrial Mid51/Fis1 complex with Fis1(WT) or Fis1(LA), revealing Fis1 species (monomer, tetramer; F) and Mid51 species (monomer, dimer, tetramer, and HMW; G), normalized to monomer levels per condition, quantified from IP immunoblot (n = 3 independent experiments). (H–J) Quantification showing coupled oligomerization of immunoprecipitated Mid51 and coimmunoprecipitated Fis1 in a Mid51/Fis1 complex. Fis1(LA) leads to normal Mid51/Fis1 binding (Fis1 IP monomer/Mid51 IP monomer ratio; H), decreased Fis1 oligomerization (Fis1 IP [tetramer/monomer ratio]; I) and decreased Mid51 oligomerization (Mid51 IP [HMW/monomer ratio]; J); quantified from IP immunoblot; n = 3 independent experiments. Mean ± SEM; unpaired two-tailed t test (A–D and H–J); ***, P < 0.001 (A, B, D, I, and J); N.S., not significant (C and H). Source data are available for this figure: SourceData F4.