In this part of the experiment, Tamm et al. transfected 293 cells with survivin and lysed them to obtain cell lysate. The lysates were incubated with different caspase forms and survivin was immunopercipitated with anti-survivin antibody. The idea behind this is that, if survivin binds physically with the caspase it is incubated with, it will be co-precipitated along with the survivin while everything else in the lysate is washed away. The immunoprecipitates were then run on SDS-PAGE and then immunoblotted for detection of the desired caspase. If the caspase of interest was detected, it meant that it was bound to survivin in the immunoprecipitation step implicating that survivin and the particular caspase had bound beforehand. Active caspase-3 and -7 coimmunoprecipitated with survivin. The inactive proforms of caspase-3 and -7 did not bind survivin. Survivin also does not bind to active caspase-8. Caspase-3 and -7 are effector proteases whereas caspase-8 is an initiator caspase that sits more upstream in the apoptotic pathway. These results demonstrate survivins capability to bind with particular caspases in vitro, but may not necessarily translate over to actual physiological conditions. Later, a 2001 study confirmed that human survivin tightly binds caspase-3 and -7 when expressed in E. coli'.
Further evidence to support the idea that survivin blocks apoptosis by directly inhibiting caspases was given by Tamm et al. 293 cells were transfected with either overexposed caspase-3 or -7 encoding plasmid and with survivin. They showed that survivin inhibited processing of these two caspases into their active forms. While survivin has been shown as mentioned above to bind to only the active forms of these caspases, it is likely here that survivin inhibits the active forms of the caspases resulting from cleaving and activating more of its own proforms. Thus, survivin acts possibly by preventing such a cascade of cleavage and activation amplification from happening resulting in decreased apoptosis.
In similar manner, looking at the mitochondrial pathway of apoptosis, cytochrome c was transiently expressed in 293 cells to look at the inhibitory effects survivin had on this pathway. Although the details are not here, survivin was shown to also inhibit cytochrome c and caspase-8-induced activation of caspases.