Additional file 1: Figure S1. of Generation and characterization of a novel transgenic mouse harboring conditional nuclear factor-kappa B/RelA knockout alleles

Whole cell lysates of A549 cells were prepared by hypotonic lysis in 50 mM NaCl, 10 mM Tris, pH 7.8, 2 mM EDTA, 1 % IGEPALCA-630, with protease inhibitor cocktail. Equal volumes of whole cell lysates were immunoprecipitated overnight with 4 μg of indicated antibody. Immunoprecipitates were captured on protein A magnetic beads and washed 4× in PBS. Samples were subjected to on-bead digestion and assayed for RelA using SID-SRM-MS [24]. Shown is mean +/- SD of RelA 756 signal relative to internal stable isotope standard (SIS). Note significant enrichment of RelA signal with each antibody. (PSD 150 kb

Rapid release of cytokines from epithelial cells cultured with ALT-E.

<p>(<b>A, B</b>) NHBE cells were cultured with PBS, ALT-E or other allergens for 15 min, and released cytokines were quantified. T. Alder, Tag Alder; S. Ragweed, Short Ragweed (n = 5 wells per treatment group). (<b>C</b>) BALF cytokine levels in naïve mice 60 min after challenge with 20 µg ALT-E or PBS control (n = 4 per group).</p

Effect of disruption of NF-κB p50 on Th2 differentiation of naïve CD4 T-cells treated with IL-18.

<p>CD4⁺ T-cells from Wild-type (WT) and NF-κB p50^−/− mice were cultured with IL-18 in the differentiation phase, restimulated with con A, and secretion of IL-4 (<b>A</b>), IL-5 (<b>B</b>) and IL-13 (<b>C</b>) was quantified by ELISA. n = 5−7 per group.</p

Effect of IL-18 on Th2 differentiation and GATA3 expression in naïve CD4+ T-cells.

<p>(<b>A,B,C</b>). Effect of NEMO peptide on Th2 differentiation. Negatively selected naïve CD3⁺ CD4⁺ T-cells were cultured with plate bound anti-CD3 and anti-CD28 with PBS (PBS), IL-18 100 ng/ml (IL-18), IL-18 100 ng/ml and 10 µM NEMO Binding Domain Binding Peptide (IL-18+NEMO) for the initial culture period. The cells were washed and restimulated, and secretion of IL-4 (<b>A</b>), IL-5 (<b>B</b>) and IFN-γ(<b>C</b>) were quantified. The data was expressed as fold increase compared to cells cultured with PBS. (<b>D,E,F</b>). GATA3 expression in T-cells. Negatively selected naïve CD3⁺ CD4⁺ T-cells were cultured with plate bound anti-CD3 and anti-CD28 in the presence of PBS (<b>D</b>), IL-4 (<b>E</b>) or IL-18 (<b>F</b>) for 7 days. GATA3 expression was measured by flow cytometric analysis.</p

Induction of epithelial cell necrosis by ALT-E.

<p>(<b>A</b>) A549 cells were cultured with ALT-E for 30 min. Changes in cell morphology were monitored by live cell imaging. (<b>B</b>) Time course of ALT-E-induced IL-18 release from airway epithelial cells. (<b>C</b>) Exposure of cultured A549 epithelial cells to ALT-E induces cell necrosis. (<b>D</b>) ALT-E challenge of mice induces sloughing of trypan blue positive necrotic airway epithelial cells (n = 4 per group). (<b>E</b>) Caspase 1 inhibitor (Cas.1 inh.) reduces IL-18 release from A549 airway epithelial cells cultured with ALT-E.</p

Effect of IL-18 on induction of Th2 differentiation.

<p>(<b>A</b>) Effect of IL-18 on IL-4 secreting splenocytes ELISPOTS. (<b>B</b>) T cell differentiation protocol used in Panels 3C–E and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030280#pone-0030280-g004" target="_blank">Fig. 4</a>. (<b>C, D and E</b>) Effect of IL-18 or IL-4 on Th2 differentiation of naïve CD4+ T-cells isolated from WT mice (<b>C</b>), IL-4^−/− mice (<b>D</b>) and WT and STAT6^−/− mice (<b>E</b>). n = 5−7 per group.</p