Figure 3

Lipopolysaccharide LPS) treatment and intermittent fasting (IF) increase the NF-κB nuclear translocation and binding activity. Electrophoretic mobility shift assay (EMSA) and Western blot were performed using nuclear extract (10 μg) from rat hippocampus. (A) Western blot results using antibodies to RELA and β-ACTIN showed that IF and LPS treatment with or without IF increased the nuclear translocation of RELA. (B) EMSA showed that LPS treatment increased the NF-κB binding activity in rats submitted or not to IF protocol. IF by itself also increased NF-κB binging activity. Competition and supershift assays of NF-κB activation by LPS treatment (C), IF (D) and IF with LPS treatment (E) were carried out. Competition studies were performed using the nuclear extract in the absence or presence of unlabeled specific (NF-κB consensus sequence, 20-fold molar excess) or nonspecific (TFIID consensus sequence, 20-fold molar excess) oligonucleotide, as indicated. Supershift assays were performed with the same nuclear extract incubated in the absence or presence of antibodies (1:20 dilution) against subunits p50, RELA, and cREL as indicated. Antibodies were added 20 minutes prior to addition of the radiolabeled NF-κB consensus oligonucleotide. The positions of specific NF-κB/DNA and non-specific (NS) binding complexes are indicated. *P < 0.05 versus Control; #P < 0.05 versus IF. N = 5 for each experimental group.