GST pull-down assay
The plasmids of human GST-eEF2, GST-eEF2 GTP mutant and GST-Drp1 were constructed as described above, and the fusion proteins of GST-eEF2, GST-eEF2 GTP mutant and GST-Drp1 were prepared as previously described. (22 ) The fusion proteins (500 μg) were incubated with GST-magnetic beads (50 μl) for 24 hours at 4 °C. After washing with 1X PBST, the GST fusion protein-conjugated beads were incubated with the extracts of HEK 293 cells transfected with eEF2 or eEF2 GTP mutant, and Drp1, for another 24 hours. The proteins retained on the beads were examined by western blot.