Fig. 6. Expression of Opa1-Q297V rendered 293T cells resistant to apoptosis.

a. GTP enhanced the in vitro self-association of Opa1. E. coli expressed and purified GST and GST-Opa1 proteins (pGEX4.1-hOpa1 variant 1, NM-015560) were mixed with radiolabeled Opa1 protein in PBS buffer supplemented with 125 mM NaCl with either 2 mM GST or GDP or without, and rotated for 1 hour in the cold room. GST and GST-Opa1 proteins were pulled down by glutathione-Sepharose beads. Beads were washed twice in PBS buffer and re-suspended in SDS buffer and run on SDS-PAGE. The presence or absence of radiolabeled Opa1 was detected by autoradiography. b–d. Expression of Opa1-Q297V rendered 293T cells resistant to BimS-induced apoptosis. 293T cells were transfected with pcDNA plasmid containing either wild-type or Q297V mutant Opa1 cDNA (tagged with flag peptide in the c-terminus) or mock transfected with empty vector. After 36–40 h, apoptosis was induced by Chariot-mediated delivery of BimS protein (see Experimental Procedures). 12 and 14 h post Chariot loading, cells were fixed and assayed for TUNEL activity (green), a measure of caspase-dependent DNA fragmentation typically produced in apoptotic cells. Nuclei are stained red by propidium iodide. The experiments were repeated three times and the percentage of TUNEL positive cells are plotted in panel c.