Drp1 GTPase assay
The plasmids of pGEX-4T-2/eEF2, pGEX-4T-2/eEF2 GTP binding mutant and
pGEX-4T-2/Drp1 were conditionally expressed in Stbl3 E. coli cells with
200 mM of isopropylthio-β-galactoside (IPTG) for 4 hours. Then, the
cells were centrifuged at 5,000 rpm for 30 min, and the pelleted cells
were suspended in nucleotide and phosphate free solution (20 mM Tris-Cl
pH 7.5 and 0.01 % Triton X-100) and sonicated. After sonication, the
cell lysates were centrifuged at 15,000 rpm for 30 min. The cell
extracts were incubated with 50 μl of GST-magnetic bead for 24 hours on
an orbital shaker at 4 °C. The GST-fusion proteins were eluted using 50
μl of elution buffer (50 mM Tris-Cl (pH 8.8), 15 mM glutathione and 1 mM
EDTA). The purified proteins, Drp1 and eEF2 or Drp1 and eEF2 GTP
deletion mutant, were mixed (vol:vol: 1 : 1= 2 μg : 2 μg) for GTPase
assay. The GTPase activity of Drp1 was measured using the colorimetric
GTPase activity assay kit (Sigma) following the manufacture’s protocol.