A. Glycerol gradient profile of Dark complexes assembled and run in PSB. The peak occurs in fractions 6–8, similar to the migration of the Apaf-1 apoptosome (not shown). Also note that Dark aggregates after heating in SDS gel loading buffer to give a triplet instead of a single band. The positions of molecular weight markers (in kDa) are shown on the left.
B. Proteolysis of DrICE in the absence and presence of Dark complexes. Lane 1: Dronc + DrICE in PSB; Lane 2: co-assembled Dark-Dronc complex + DrICE in PSB; p20 and p10 are cleavage products of DrICE.
C. Frozen-hydrated Dark double-rings at 3 mg/ml in PSB imaged over a hole in the carbon support film.
D. Dark double-rings assembled at 0.5 mg/ml in PSB and imaged on a thin carbon film
E. Dark-Dronc complexes were assembled in PSB and imaged over holes at 3 mg/ml. The images show mostly single-ring aggregates as evidenced by the lack of typical side views for the double-ring and the presence of single-ring edge views.