il\VOLVEMENT OF RETII\OIC ACID II{ - MSpace at the University of ...

polyclonal antibody (Signa-Aldrich CO, St. Louis MO, USA). Primary antibody was
detected using a goat anti-rabbit IgG horseradish peroxidase conjugated secondary
antibody (Bio-Rad, Hercules, CA, USA). Molecular weights of the separated proteins
were determined using a standard (Bio-Rad, Hercules, CA, USA) and biotynilated (Cell
Signaling Technology inc., Beverly, MA, USA) protein ladder molecular weight markers.
The detection of membrane-bound proteins was performed using the BM
Chemiluminiscence (POD) western blotting system (Roche Diagnostics GmbH,
Manheim, Germany). The bands were visualized using a Flour S-Multi-imager MAX
system (Bio-Rad, Hercules, CA, USA) and quantified by an image analysis software
(Quantity One, Bio-Rad, Hercules, CA, USA).
Il.d.Annexin-Propidium Iodide Assav
Occurrence of apoptosis in isolated cardiac myocytes was detected using a
commercially available Armexin-V-FLUOS assay kit (Roche Diagnostics GmbH,
Mannheim, Germany)(van Heerde et al. 2000). After the initial treatment with retinoic
acid and adriamycin, isolated adult myocytes were washed with PBS. Immediately after
the washing, cells were exposed to 20 pl of Annexin-V-FLUOS staining solution and 20
pl of propidium iodide in a total volume of 250 pl of PBS per dish. The cells, protected
from light, were incubated in humidified chamber for 30 minutes at l5-25o C. After the
incubation, samples were washed twice with phosphate buffered saline (PBS). The cells
were mounted for microscopy using a Floursave reagent (Calbiochem, San Diego, CA,
USA). The rod shaped myocytes exhibiting the green fluorescence (Annexin-V-FLUOS)
were counted as the ones in the early apoptosis. The cells exhibiting no fluorescence at all
were counted as the normal ones. Rounded myocytes showing red nuciei stained with
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