Anti creb sc 186

All reagents were of analytical grade. DMEM media and fetal calf serum were purchased from GIBCO Life Technologies (Grand Island, NY). Insulin, dexamethasone, 3-isobutyl-methyl-xanthine, Oil Red O, EtOH Triton-X100, BriJ, and protease inhibitor were purchased from Sigma-Aldrich (St. Louis, MO). Recombinant human irisin was purchased from (Cayman Chemical, Michigan, USA). Anti-phospho-NF-κB [sc101748; working dilution (wd) 1:1000], anti-NF-κB (sc109, wd 1:1000), anti-AKT (sc 1619, wd 1:1000), anti UCP-(sc 6529, wd 1:1000), anti-CREB (sc 186, wd 1:1000) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-ERK1,2 (#4695, wd 1:2000), anti-phospho-AKT (#4060, wd 1:2000) and anti-phospho-ERK1/2 (#4370, wd 1:2000) antibodies were purchased from Cell Signaling Technology (Beverly, MA). Donkey HRP-labeled secondary antibodies (anti-mouse Cat #SA1-100, wd 1:5000; anti rabbit Cat #31458, wd 1:5000; anti goat Cat #A16005, wd 1:5000) were purchased from Thermo Fisher Scientific.

Total proteins extracted from hippocampus were subjected to SDS-PAGE on 12% separating gels and electrophoretically transferred onto nitrocellulose membranes. After blocking with 5% nonfat dry milk, the membranes were incubated with the primary anti-CREB (sc-186, Santa Cruz Biotechnology) or anti-BDNF (sc-20981, Santa Cruz Biotechnology) antibody at 4°C overnight, followed by a corresponding horseradish peroxidase conjugated secondary antibody (Sigma-Aldrich) for 1 h at room temperature. The reaction was visualized by TMB staining. Densitometric signals were normalized to internal standard β-actin and analyzed with Bio 1D software.