Activation of MAPK and NF-κB pathways in samples was determined as the ratio between the levels of phosphorylated (phospho-) and total ERK1/2, p38, and JNK kinases and those of NF-κB inhibitor IκB-α, for the MAPK and NF-κB pathway, respectively.
Molecule phospho- and total levels were analyzed by multiplex immunobead-based assay (Cell Signaling assay, Upstate Beadlyte, Dundee, UK) based on biological testing technology (Luminex xMAP; Luminex Corp., Austin, TX), according to the manufacturer's protocol. Briefly, tissues were homogenized in 300 μL lysis buffer B from the kit supplemented with protease inhibitors (Complete Mini Protease Inhibitor Cocktail, Roche Applied Science, Mannheim, Germany) and phosphatase inhibitor (1 mM sodium orthovanadate; Sigma-Aldrich Quimica SA Madrid, Spain). Homogenates were gently agitated for 15 minutes at room temperature and then centrifuged at 6000 rpm for 4 minutes Supernatants were collected and stored at −80°C until assayed. Total protein concentration in supernatants was determined by the BCA protein assay (Pierce, Rockford, IL), and 2 μg of total protein (25 μL) of each sample was pipetted into assay plate wells. To this was added a 25 μL mixture of beads coupled to phospho-ERK1/2–, phospho-p38–, phospho-JNK–, and phospho-IκB-α–specific capture antibodies (Upstate Beadlyte, Dundee, UK). A separate assay was run with a 25 μL mixture of beads coupled to total ERK1/2-, total p38-, total JNK-, and total IκB-α–specific capture antibodies (Upstate Beadlyte). After overnight incubation under agitation at 4°C in the dark, the beads were washed and mixed with biotinylated specific reporter antibodies for 1 hour at room temperature, followed by washing and subsequent incubation with streptavidin-phycoerythrin, for 30 minutes. The beads were finally washed and resuspended in 100 μL assay buffer, and the amounts of phospho- and total JNK, ERK1/2, and p38 kinases, and IκB-α (determined as the median fluorescence intensity in arbitrary units for each molecule in each sample) were measured (Luminex 100 IS 2.3 system; Luminex, Austin, TX). Samples were analyzed in duplicate.