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Fig. 5. (A) Analysis of mRNA and protein from aerobic and hypoxic (24 h) cardiac
myocytes. Parallel plates of cardiac myocytes were cultured under aerobic or
hypoxic conditions for 24 h and then harvested for RNA extraction. RNA was
purified and reverse transcribed by standard procedures. Differentially
labeled cDNAs were hybridized to arrays of 20 000 specific gene sequences
(known and unknown cDNAs and expressed sequence tags) from combined rat and
mouse libraries (Incyte Inc., Freemont, CA, USA). Data were analyzed using
Excel 98 software. Hypoxia-regulated marker genes including heme oxygenase
(HO), glucose transporter (GLUT), pyruvate dehydrogenase kinase (PDK),
triosephosphate isomerase (TPI), tyrosine amino transferase (TAT) and
metallothionein (MT-1) are shown. (B) Northern blot of cardiac myocyte RNA
extracted from hypoxic cultures. The top gel shows BNIP3; the bottom gel shows
ß-actin. (C) Western blot analysis of proteins from hypoxic cardiac
myocytes as in A. Anti-BNIP3 recognizes two bands at 
60 kDa and 30 kDa,
corresponding to SDS-resistant homodimers and monomers, respectively. Lower
panels show the same blot probed with anti-Bax, Bak and ß-actin. (D) Rat
hearts were removed and perfused by the Langendorf method, as described
previously (Webster et al.,
1999). Hearts were subjected to no flow for 1 h or to perfusate
equilibrated with 100% N2 for 2 h. RNA was analyzed by northern
blot, as above.
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