2.6. Electrophoretic Mobility Shift Assay (EMSA)
Nuclear proteins were isolated from brain tissue. Briefly, after removal of supernatant (cytoplasmic extract), the pellets were resuspended in nuclear extraction reagent. The tubes containing pellets were put on vortex for 15 seconds every 10 minutes, for a total of 40 minutes. The tubes were centrifuged at maximum speed (~16,000 × g) in a micro centrifuge for 10 minutes. The supernatants (nuclear extract) were transferred to a clean prechilled tube and stored at −80°C until use. EMSA was performed as described previously [8]. NFκB binding activity was examined in a 15 μL binding reaction mixture containing 15 μg of nuclear proteins and 35 fmol [γ-32P] labeled double-stranded NFκB consensus oligonucleotide. A super shift assay using antibodies to P65 and P50 was performed to confirm NFκB binding specificity as previously described.