Expression of cardiac vasodilator specific phosphoprotein.
ischemia
HYPOXIC, ISCHEMIA
In separate experiments, isolated and perfused mouse hearts were subjected to 5 minutes of ischemia following 30 minutes of stabilization. The supernatant from hypoxic or normoxic mouse RBCs was given to the isolated heart at the start of ischemia. After 1 minute reperfusion, the hearts were harvested using liquid nitrogen and stored at –80°C. Protein was extracted from the heart by using radioimmunoprecipitation assay buffer (RIPA buffer, VWR International) containing protease inhibitors (Roche Diagnostics). The protocol of Western blotting was described previously (To further localize the expression of pVASP and determine the role of cGMP, additional hearts were collected for immunofluorescence using a protocol described elsewhere (