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.