4.10. Western Blot Analysis for IκB-α, NF-κB, COX-2, and iNOS

Cytosolic and nuclear extracts of gingivomucosal tissues were prepared as previously described by Reference [48].

In the cytosolic fraction, the expressions of kappa light polypeptide gene enhancer in B cells inhibitor alpha (IκB-α), iNOS, and cyclooxygenase 2 (COX-2) were quantified.

In the nuclear fraction, the expression of NF-κB was quantified. Filters were blocked with 1× PBS, 5% (w/v) nonfat dried milk (PM), for 40 min, at room temperature, and then probed with following antibodies: anti-IkB-α (1:500, Santa Cruz Biotechnology, Dallas, Texas, USA #sc1643), anti-NF-κB (1:500, Santa Cruz Biotechnology, #sc8008), anti-Cox2 (1:500, Santa Cruz Biotechnology, #sc-1746), anti-iNOS (1:500, Santa Cruz Biotechnology, #sc8310) in 1× PBS, and 0.1% Tween-20, 5% w/v nonfat dried milk (PMT) at 4 °C, overnight. After that, the membranes were incubated with peroxidase-conjugated bovine anti-mouse IgG secondary antibody or peroxidase-conjugated goat anti-rabbit IgG (1:2000, Jackson ImmunoResearch, West Grove, Pennsylvania, USA) for 1 h, at room temperature. To ascertain that blots were loaded with equal amounts of proteins, they were also incubated in the presence of the antibody against GAPDH (cytosolic fraction 1:500; Santa Cruz Biotechnology) or lamin A/C (nuclear fraction 1:500 Sigma-Aldrich Corp.), as described by Reference [49].