Modulation of Pineal Melatonin Synthesis by Glutamate Involves Paracrine Interactions between Pinealocytes and Astrocytes through NF-B Activation
Electrophoretic mobility shift assays for NFκB in the pineal gland. (a) Competition studies were performed using nuclear extract (5 μg) from cultured pineal glands treated with glutamate (Glu) 100 and 600 μM, in the absence or presence of unlabeled specific (NF-κB consensus sequence, 10-fold molar excess) or nonspecific oligonucleotide (TFIID consensus sequence, 10-fold molar excess), as indicated. (b, c) Supershift assays were performed with the same nuclear extract (5 μg) incubated in the absence or presence of antibodies against the subunits p50 (1 : 10 dilution), p65 (1 : 20 dilution), p52, and c-Rel (1 : 10 dilution), as indicated. Antibodies were added 20 min prior to the addition of the radiolabeled NF-κB consensus oligonucleotide. The position of specific NF-κB/DNA binding complexes p50/p50 (band 1, b, c) is indicated. NS represents no specific binding (bands 2 and 3, a, b, c). The localization of the free probe is also indicated.