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Il.c.Western Blot Analvsis
Western blot analysis was performed on isolated cardiac myocytes using a
previously described method (Sano et al. 2003). Control and treated isolated cardiac
myocytes were washed with PBS. Cells were then mechanically lifted from the dish
bottom using a cell scraper and then suspended in PBS and centrifuged at 1000 rpm for
10 minutes. Supernatant was discarded and the pallet was re-suspended in the cell lysis
medium containing the RIPA buffer (150 mM NaCl, 1% NPaO, 0.5% deoxycholic acid,
0.1% SDS, 50mM Tris) and Sigma-Aldrich Protease inhibitor cocktail for mammalian
tissues consisting of AEBSF, 100; Aprotinin, 0.08; Leupeptin, 2.2; Bestatin, 4.0;
Pepstatin A, 1.5; and E-64 144 in mM. Re-suspended pallets were sonicated, frozen in
liquid nitrogen and stored at -75oC. The protein samples were then subjected to onedimensional
sodium dodecyl suplhate polyacrylamide gel electrophoresis (SDS-pAGE)
in a discontinuous system following a previously described method (Laemmli lg70). 5%
gel was used for protein stacking phase while 15% gel was used for the separation
analysis of isolated proteins. Separated proteins were then transferred onto 0.45 ¡rm
nitrocellulose membrane using a transfer buffer which consisted of 20mM Tris, 150 mM
glycine, 20o/o methanol and 0.02% SDS. The nonspecific binding sites were blocked by
overnight incubation with 5o/o nonfat milk in Tris-buffered saline/O.l% Tween 20
solution. After the blocking, the membranes were processed for immunodetection using
rabbit specific IgG RAR (o,Þ,y) and RXR (o,F,T) polyclonal antibodies (Santa Cruz,
Santa Cruz, CA, USA). Apoptotic proteins, BAX and BCL-xl, were also detected using a
rabbit IgG BAX and BCL-xl polyclonal antibodies (Cell Signaling Technology inc.,
Beverly, MA, USA). PPAR ô protein levels were detected using a rabbit IgG ppAR ô
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