Western Blot Analysis of NF-κB Signaling Pathway

Proteins (50 μg) from all sample groups were processed as previously described [40 (link)]. Sheets were incubated 12 h at 4°C in with primary antibodies to NFκB (1 : 500, Santa Cruz Biotechnology), MyD88 (1 : 1000, ThermoFisher Scientific), p300 (1 : 750, EpiGentek), DNMT1 (1 : 750, OriGene), and β-actin (1 : 1000, Santa Cruz Biotechnology) [41 (link), 42 (link)]. Then, sheets were maintained at room temperature for 30 min with peroxidase-conjugated secondary antibody diluted 1 : 1000 in 1x TBS, 5% milk, and 0.05% Tween-20 [43 (link)]. The ECL method was used for band visualization, and the protein level were measured by means of the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA) [44 (link)].