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.