Figure 4
Administration of db-cAMP suppresses caspase-3 activation and activity in cardiac allografts. (A) Transplanted hearts were harvested and homogenized in a 5-fold volume of lysis buffer. Tissue extract was obtained by centrifugation at 100,000 × g for 20 min at 4℃. Caspase-3 activity was assayed by colorimetric assay using the chromogenic substrate Ac-DEVD-pNA. Data shown are the mean ± SD (n = 4). *P < 0.05; **P < 0.01 versus non-treated allograft. (B) Transplanted hearts were harvested on POD 5, and the tissue extracts were prepared by homogenizing and centrifuging at 100,000 × g for 20 min at 4℃. The tissue extract (10 mg) was incubated with 35S-labelled PARP in a final volume of 15 ml at 37℃ for 1 h and then mixed with an equal volume of SDS-sample buffer. PARP cleavage activity was determined by gel electrophoresis and autoradiography. (C) Tissue extracts (40 µg protein) from transplanted hearts were separated on 14% SDS-PAGE and transferred onto a nitrocellulose membrane. Caspase-3 activation was determined by Western blot analysis using a polyclonal antibody for caspase-3.
