Skip to main content

Advertisement

Fig. 2 | Cell & Bioscience

Fig. 2

From: Dimerized translationally controlled tumor protein increases interleukin-8 expression through MAPK and NF-κB pathways in a human bronchial epithelial cell line

Fig. 2

The role of NF-κB and AP-1 in dTCTP-induced IL-8 production. Electrophoretic mobility shift assays to measure the DNA-binding activities of NF-κB (a) and AP-1 (b) binding to their recognition sites were performed. Nuclear extracts were prepared from the BEAS-2B cells stimulated with 10 μg/ml of dTCTP for various time periods (0, 30 and 60 min). EMSA was performed using nuclear extract binding buffer (100 mm Tris, 500 mm KCl and 10 mm DTT; pH 7.5), and biotinylated probes were incubated at room temperature for 20 min. The protein–DNA complexes were electrophoresed on 6% polyacrylamide gels at 4 °C in ×0.5 TBE buffer and transferred to a nylon membrane. The UV cross-linked membrane was treated with streptavidin–horseradish peroxidase conjugate and then detected with CCD camera imaging device using the LightShift Chemiluminescent EMSA Kit. Unlabelled oligonucleotides were included to examine binding specificity. c Schematic presentation of IL-8 promoter luciferase reporter system and two mutant IL-8 promoters with an altered NF-κB or AP-1 binding site (left panel). Luciferase activities of BEAS-2B cells carrying IL-8 promoter reporter plasmids were measured using dual luciferase assay system. BEAS-2B cells were transiently transfected by Lipofectamine™ 2000 reagent with 1 μg of each IL-8 luciferase plasmids and 0.1 μg of pRL-TK. At 24 h after transfection, the cells were stimulated for 24 h with dTCTP (10 μg/ml). Values are expressed as fold changes (mean ± SEM, n = 3). **p < 0.01, ***p < 0.001; control vs dTCTP, p < 0.05, ††p < 0.01; dTCTP treated wild type vs mutant

Back to article page