Cleavage of the PAR-3 N-terminal end by aPC is required, but not sufficient, to inhibit apoptosis. (A) Schematic representation of the V5-tagged wild-type and V5-tagged mutant PAR-3 expression constructs. (B) Representative image showing the expression of V5-tagged PAR-3 wild-type (PAR-wt–V5) and V5-tagged mutant expression constructs (PAR-m–V5, PAR-3–TEM-1–V5) in transiently transfected mesangial cells (semiquantitative RT-PCR). (C) Representative immunoblot showing V5 levels in the culture supernatant after treatment with PBS or aPC (20nM) for 1 hour of cells transfected with V5-tagged PAR-3 wild-type and V5-tagged mutant PAR-3 expression constructs. The detection of the V5 epitope in the supernatant reflects proteolytic activation of PAR-3. (D) Bar graph summarizing the frequency of apoptosis in staurosporine-treated mesangial cells transfected with V5-tagged PAR-3 wild-type and V5-tagged mutant PAR-3 expression constructs. Only full-length PAR-3 with a nonmutated cleavage site enables aPC to inhibit apoptosis. Data are mean ± SEM of at least 3 independent experiments. *P < .01 vs control (ANOVA).