Amino acid substitutions within the PECAM-1 extracellular homophilic binding domain, or within its cytoplasmic ITIM, abolish PECAM-1—mediated cytoprotection. The 293 cells were transiently transfected with plasmids encoding the indicated proteins. Twenty-four hours after transfection, cells were either stained for apoptotic nuclei (A) or used to prepare detergent lysates for caspase activity measurements (B), which were carried out essentially as described in the legend to Figure 1. While PECAM-1 effectively suppressed Bax-mediated cell death, expression of an equivalent amount (see the characteristic PECAM-1 doublets in the anti—PECAM-1 immunoblot) of either of 2 mutated forms of PECAM-1, the first differing in only 2 Tyr→Phe amino acid substitutions within PECAM-1's cytoplasmic ITIM and the second containing a single Lys89Ala amino acid substitution within the PECAM-1 extracellular homophilic binding domain, conveyed no cytoprotective effect at all. n represents the number of times each experimental condition was repeated. P values were derived using a paired Student t test. The y-axis (“relative caspase activity”) represents normalized data from up to 11 different experiments, and the data are presented as percentage of caspase activity present in the Bax overexpression—induced control. * and ** indicate P values compared with vector-transfected cells.