FIGURE 7.

Inhibition of C9 polymerization by the ATPase domain of mortalin. A, C9 was mixed with recombinant full-length mortalin, mortalin C-terminal SBD, mortalin N-terminal ATPase domain, or BSA as control for 15 min at 37 °C and then incubated with 42 or 100 μm ZnCl₂ for 2 h at 37 °C. The samples were subjected to SDS-PAGE on a 3–10% acrylamide gradient gel and stained with Coomassie Blue. The bands of polyC9, monomeric C9, mortalin, SBD, and ATPase domain are indicated; the bands of C9 and BSA did not separate. B, C9 was mixed with recombinant full-length mortalin, mortalin-V482F (mV482F), or BSA as control for 15 min at 37 °C and then incubated with 42 or 100 mm ZnCl₂ for 2 h at 37 °C. C9 polymerization was analyzed as described in A. C, mortalin or mV482F was attached to microtiter plate wells and then incubated with C9 for 1 h in 37 °C. The wells were then washed, treated with anti-C9 antibody and peroxidase-labeled second antibody, developed with OPD, and analyzed in an ELISA reader.