Journal: Experimental cell research

Article Title: Inhibition of inducible NF-kappaB activity reduces chemoresistance to 5-fluorouracil in human stomach cancer cell line.

doi: 10.1016/s0014-4827(03)00223-4

Figure Lengend Snippet: Fig. 3. NF-B DNA-binding activity and transcriptional activity in 5-FU-treated cells. (A) EMSA. Cells were treated with 10 M 5-FU for 5, 10, or 20 h as indicated. Both cell lines had constitutive NF-B activation, although inducible activation of NF-B was detected only in NUGC3/5FU/L cells after 10 h of 5-FU treatment. Control represents untreated cells. The results are representative of three separate experiments. (B) Supershift assay. Preincubation of nuclear extracts from NUGC3/5FU/L cells treated or untreated with anti-p65, p50, or cRel antibody showed that p65 and p50 were the predominant subunits of inducible NF-B after 10 h treatment of 10 M 5-FU. (C) Transcriptional activity of NF-B measured by luciferase reporter gene assay. Cells were transfected with the NF-B promoter/luciferase construct and then treated with 10 M 5-FU for 5, 10, or 20 h, followed by examination of luciferase activity. Control represents untreated cells. The Promega Dual-Luciferase Reporter Assay system was used according to the protocol provided by the manufacturer. Sample luciferase activity was normalized to control luciferase activity. Increased luciferase activity was detected in NUGC3/5FU/L but not in NUGC3 cells after 10 h of treatment with 5-FU (mean SD of three independent experiments).

Article Snippet: Identification of the distribution of primary antibody (antiNF- B p65 antibody; Santa Cruz Biotechnology, Santa Cruz, CA) was achieved by subsequent application of a biotinylated anti-primary and streptavidin-peroxidase.

Techniques: Binding Assay, Activity Assay, Activation Assay, Control, Luciferase, Reporter Gene Assay, Transfection, Construct, Reporter Assay