Goat anti rabbit or anti mouse peroxidase conjugated secondary antibody

Primary astrocytes were pretreated with leptin for 1 h and then treated with 10 mM glutamate for 1 h. Cell lysates were prepared with lysis buffer (10 mM Na 2 HPO 4 (pH7.2), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% sodium deoxyclolate) supplemented with Xpert protease inhibitor cocktail (GenDEPOT, Inc., Baker, TX, USA). Total proteins were resolved on 12% SDS-polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (PVDF) (Amersham Pharmacia Biotech, Uppsala, Sweden). Membranes were blocked in 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature and then incubated with anti-ERK1/2, anti-phospho-ERK1/2 (Cell Signaling Technologies), anti-p38, anti-phospho-p38, anti-JNK, anti-phospho-JNK, and anti--tubulin antibodies overnight at 4°C. After three washes with 0.1% TBST, blots were incubated with goat anti-rabbit or anti-mouse peroxidaseconjugated secondary antibody (Santa Cruz Biotechnology) for 2 h and washed with 0.1% TBST. Western blot bands were developed by the enhanced chemiluminescence (ECL) method (GE healthcare, Buckinghamshire, UK).