Fig. 2: Pairwise interactions between SDHA, SDHAF2, and SDHAF4.
The interaction of His-SDHA (2.6 µM) with SDHAF2 (6.4 µM) and SDHAF4 (8 µM) was evaluated by using a Ni-NTA pull-down assay. SDHA was tested in several of its forms: apo-SDHA, SDHA with bound non-covalent FAD, and holo-SDHA with covalently attached FAD. His6-SDHA was incubated with purified SDHAF2 and SDHAF4 as indicated and associated proteins were evaluated by SDS-PAGE. FAD was added at 75 µM and fumarate was added at 5 mM. Yellowish FAD fluorescence is observed when SDHA is covalently attached to FAD. ImageJ densitometry, shown at the bottom, was measured as arbitrary units (arb. units.) and used to evaluate the relative binding of SDHAF2 (teal) and SDHAF4 (brown) to SDHA. The y-axis on the densitometry quantitation expresses these as a ratio. a Input protein and pairwise interaction between SDHA and SDHAF2. (left) input SDHA, (right) interaction between SDHA and SDHAF2 in the presence of FAD and fumarate. Note that only after the addition of fumarate does the covalent bond between FAD and SDHA form (lane 3). b Pairwise interaction of SDHA and SDHAF4. c Interaction of SDHA with the assembly factors after incubation with both SDHAF2 and SDHAF4. d Displacement of SDHAF2 after purified holo-SDHA/SDHAF2 complex (2 µM) was incubated with SDHAF4. All Coomassie gels are representative of n = 4 independent experiments, bar graphs show mean values ± SD, and statistics were done by paired two-tailed Student’s t-test. Source data are provided as a Source Data file.
