Introduction 2007 2003 2006 2005 2005 1999 2007 2000 2003 2001 2001 2001 2001 1999 2005 2000 2005 2006 2005 2007 Materials and methods Animals and diet protocol, 40% caloric restriction for 6 months R R R n n n n RNA extraction, RT-reaction and quantification by PCR 2007 Real time PCR ® ® Western blot Liver tissue was rapidly homogenized in a buffer containing 50 mmol/l Tris·HCl, 1% SDS, 1 mmol/l sodium-orthovanadate, 5 mmol/l EGTA, 1 mmol/l PMSF, 1 μg/ml aprotinin, and 1 μg/ml leupeptin. Proteins were quantified using the bicinchoninic acid protein assay (Pierce). Protein (50 μg) in 2× Laemmli SDS sample buffer were boiled for 5 min and after centrifugation loaded onto a SDS-PAGE gel. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 100 V for 90 min. The filters were blocked with 0.01% Tween, 2% nonfat milk, and then incubated with antibodies directed against MIF (Abcam) and GAPDH (Abcam). Statistical analysis P Results Prevention of age-dependent increase of PHD3 in heart, liver and skeletal muscle 1 2 3 Fig. 1 Effect of age and caloric restriction on PHD1 mRNA expression. Tissue samples (heart, liver and skeletal muscle (M. gastrocnemicus)) were obtained from young (6 months old) and old (24 months old) rats with or without a 40% caloric restriction applied for 6 months. Subsequently, RNA was extracted and PHD1 mRNA quantitated by real time PCR  Fig. 2 Effect of age and caloric restriction on PHD2 mRNA expression. Tissue samples (heart, liver and skeletal muscle (M. gastrocnemicus)) were obtained from young (6 months old) and old (24 months old) rats with or without a 40% caloric restriction applied for 6 months. Subsequently, RNA was extracted and PHD2 mRNA quantitated by real time PCR  Fig. 3 Effect of age and caloric restriction on PHD3 mRNA expression. Tissue samples (heart, liver and skeletal muscle (M. gastrocnemicus)) were obtained from young (6 months old) and old (24 months old) rats with or without a 40% caloric restriction applied for 6 months. Subsequently, RNA was extracted and PHD3 mRNA quantitated by real time PCR  2006a 1 2 3 Caloric restriction affects the expression of the macrophage migration inhibitory factor 2005 2006 4 5 4 Fig. 4 Effect of age and caloric restriction on MIF mRNA expression. Tissue samples (heart, liver and skeletal muscle (M. gastrocnemicus)) were obtained from young (6 months old) and old (24 months old) rats with or without a 40% caloric restriction applied for 6 months. Subsequently, RNA was extracted and MIF mRNA quantitated by real time PCR  Fig. 5 Effect of age and caloric restriction on MIF protein levels. Liver tissue samples were obtained from young (6 months old) and old (24 months old) rats with or without a 40% caloric restriction applied for 6 months. Subsequently protein was extracted and MIF and GAPDH protein levels were investigated by immunoblot analysis  Discussion 2000 2005 2004 2004 2004 2006 2002 2005 2002 2006 2007 2006 2005 1999 2000 2005 2007 2007 2005 2007 2006 2006 2006