Immunoprecipitation
HEK293 cells were transfected with pcDNA, pcDNA eEF2, pcDNA eEF2 GTP
mutant, and pGW1-Drp1 using lipofectamin 3000 reagent (Invitrogen) for 3
days according to the manufacturer’s protocol. The eEF2 and Drp1
antibodies were conjugated with protein A/G agarose bead for overnight
at 4 °C. Anti-IgG (Rabbit) used as a control (Santa Cruz
biotechnologies). The beads were washed three time in ice cold 1X TBST
and incubated with 500 μg whole cell extracts of pcDNA, eEF2, eEF2 GTP
mutant, and Drp1 for 24hrs at 4 °C. After washing by 1X TBST three
times, antibody-conjugated proteins were retained from the beads using
SDS-sample buffer (Bio-Rad) and were analyzed by western blot.