Figure 4. Binding of purified ETFB to GCDH.

ETFB-His₆ expressed and purified from E. coli was immobilized on Ni-NTA agarose and incubated with extracts from BHK cells overexpressing GCDH-Myc (ETFB+GCDH) for 2 h. Cell extracts overexpressing LC3-GFP were used as negative control (ETFB+LC3). Aliquots of cell extract (input, I: 10% of total), the unbound protein supernatant after precipitation of ETFB-Ni-NTA beads (S: 10%), last wash (W, 25%) and the eluted fraction (E, 100%), containing the bound proteins were separated by SDS-PAGE (10% acrylamide) and analyzed by anti-GCDH and anti-LC3 immunoblotting. The expression of ETFB used for pull-down was analyzed by anti-ETFB western blotting. The image shows representative blots of n = 5 independent experiments.