Anti erk

Frozen muscle strips or pieces of the left ventricle were thawed on ice in 50 μL of homogenization buffer [1% (v/v) NP 40 (IGEPAL CA‐630), 10% (v/v) glycerol, 137 mM NaCl, 20 mM Tris–HCl pH 7,4, 20 mM NaF, 1 mM Na‐orthovanadate, 1 mM Na‐pyrophosphate, 50 mM β‐glycerophosphate, 10 mM ethylene‐diamine‐tetra‐acetic acid (EDTA) pH 8, 1 mM ethylene glycol‐bis‐(2‐aminoethyl)‐tetra‐acetic acid (EGTA) pH 7, 4 μg/mL aprotinin, 4 μg/mL leupeptin, 4 μg/mL pepstatin A, 1 mM phenylmethanesulfonyl fluoride (PMSF)] and homogenized. Protein of the suspensions was determined with BCA™ Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA) and 20 μg of samples subjected to SDS‐PAGE. Western blotting was carried out according to standard protocols. The following antibodies were used: anti‐P‐AKT, anti‐AKT, anti‐P‐GSK3β, anti‐P‐p38, anti‐P‐ERK, anti‐ERK, anti‐P‐JNK (New England Biolabs, Ipswich, MA, USA), anti‐GAPDH (BioTrend, Cologne, Germany), anti‐mouse IgG, horseradish peroxidase‐linked, and anti‐rabbit IgG horseradish peroxidase‐linked (Amersham Biosciences, Freiburg).
For quantification, an enhanced chemo luminescence detection system (Amersham) was used according to the manufacturer's instructions.