Modulation of NF-kB and p38 MAPK activation in gingival tissues by systemic administration of curcumin. The animals were sacrificed after 15 days of curcumin (30 and 100 mg/Kg) or vehicle administration and the tissues surrounding the lower first molars were harvested. Total protein was isolated from gingival biopsies with a detergent-containing buffer (T-Per, Pierce) supplemented with protease and phosphatase inhibitor cocktails (Complete and Phos-Stop, Roche). Protein concentration was determined with a Lowry-based microassay (DC assay, Bio-Rad) and 60 μg of each sample were diluted in LDS-containing running buffer supplemented with DTT, heat-denatured, electrophoresed in 10% Tris-Acrilamide gels and transferred to 0.2 μM nitrocellulose membranes in a semi-dry transfer apparatus. The activation of the signaling pathways was assessed by the detection of phosphorylated forms of p65 (NF-kB) and p38 MAPKinase. Expression levels of constitutive housekeeping GAPDH are shown to confirm equal protein loading. The images are representative of samples from three different animals in each experimental group.