EKB Radiosensitizes Squamous Cell Carcinoma Figure 1. Effect of EKB-569 on radiation modulated NFkB, AP1 and SP1 DNA binding activity. A representative autoradiograms showing the NFkB-DNA binding activity in the nuclear extracts of human SCC-4 cells (A) or SCC-9 cells (B) that are either treated with EKB-569 alone (upper panel) or in combination with IR (Lower panel). NF-kB-specific bands are indicated by an arrow head. Autoradiogram was slightly overexposed to reveal EKB-inhibited NF-kB-specific bands. Densitometric analysis of three independent experiments showing dose-dependent inhibition of NFkB- DNA binding activity in SCC-4 cells (C) and SCC-9 cells (D). (E) Time-dependent inhibition of NFkB-DNA binding activity in human SCC-4 cells by EKB- 569 (5.0 mg) in the presence or absence of IR exposure. EMSA was carried out in the nuclear extract at 1, 3, 6, 24, 48 and 72 h post-exposure. (F) Representative autoradiogram from three independent experiments showing AP-1 DNA binding activity in SCC-4 cells treated with EKB-569 (1.0, 2.0 and 5.0 mg) or exposed to IR in the presence or absence of EKB-569. (G) Representative autoradiogram from three independent experiments showing SP-1 DNA binding activity in SCC-4 cells treated with EKB-569 (0.5, 1.0, 2.0 and 5.0 mg) or exposed to IR in the presence or absence of EKB-569. doi:10.1371/journal.pone.0029705.g001 competition binding assay was performed. The NFkB DNA- binding activity was competitively reduced to 47% and 36.4% by the addition of 0.02 and 0.2 pmol of homologous unlabeled NFkB specific-double stranded oligonucleotide, respectively. Supershift analysis with p50 and p65 antibodies further confirmed that the gel shifted bands are indeed NFkB (data not shown). PLoS ONE | www.plosone.org 4 December 2011 | Volume 6 | Issue 12 | e29705