Fig. 4

EMSA identification of physical interaction of NF-κB with the predicted binding sites of NOX1 promoter. a Schematic drawing of two putative NF-κB binding sites in the proximal promoter of human NOX1 genes based on the bioinformatic analysis. Lane 1: -1095/-1086 bp (NOX1/κB1); Lane 2: -261/-252 bp (NOX1/κB2). b EMSA was performed to analyze whether the nuclear protein was able to recognize and bind to the two putative NF-κB binding sites in the promoter of NOX1. Both Lane1 and Lane2 contained the nuclear protein extracted from A549 treated with TNF-α (10 ng/ml) 24 h and different probes. Lane1: probe based on NOX1/κB1; Lane2: probe based on NOX1/κB2. Experiments were run in triplicate and repeated 3 times. c EMSA was performed to confirm whether NF‐κB was able to recognize and bind to the putative NOX1/κB2 region of the NOX1 promoter. Nuclear protein was extracted from A549 treated with TNF-α (10 ng/ml) 24 h. Lane 1: contain no nuclear protein; Lane 2: contain both nuclear protein and probe 2; Lane 3: contain both nuclear protein and probe 2, an additional 100-fold molar excess of unlabeled probe 2; Lane 4: contain both nuclear protein and probe 2, an additional 100-fold molar excess of unlabeled probe 2 with scrambled sequences; Lane 5: contain both nuclear protein and probe 2, the nuclear protein was incubated with anti-NF-κB/p65 antibody predictably for 15 min. Experiments were run in triplicate and repeated 3 times