Seladin-1 is a substrate for caspases in tissue culture and in vitro. A, Confluent HUVECs were deprived of growth factors for 12 hr. Cell lysates of control cells, of surviving viable cells, and of apoptotic floaters were immunoblotted and analyzed with a specific, antigen affinity-purified, antiserum against seladin-1. B, Confluent HUVECs were deprived of growth factors for 16 hr in the absence (lane 1) or presence (lane 2) of ZVAD. Cell lysates including attached and floating cells were immunoblotted and analyzed with the seladin-1 antibody. C, In vitrotranslated and [35S]-methionine-labeled seladin-1 was incubated at 37°C for 1 hr and 2.5 hr with reaction buffer alone (control) or with recombinant caspase-3, caspase-6, or caspase-7. The samples were analyzed on 10% SDS-PAGE, and full-length seladin-1 as well as seladin-1 fragments 1–4 were visualized by autoradiography.
