Fig. 5.

PKCε is a new downstream target of NF-κB. a, b H1299 cells were transiently transfected with p65 siRNAs. Forty-eight hours later, total RNAs were extracted. The mRNA levels of p65 (a) and PKCε (b) were determined by q-PCR. Data represent the average of three independent experiments (mean ± SD). ***P < 0.001. c H1299 cells were transiently transfected with p65 siRNAs. Forty-eight hours later, the cells were lysed. Protein expression was assessed by western blotting using the indicated antibodies. d Schematic depiction of the NF-κB binding sequence in the promoter region of PKCε. e ChIP assay was conducted with anti-NF-κB (p65), anti-AcH3 antibodies, or control rabbit IgG for immunoprecipitation, followed by PCR with PKCε promoter-specific and GAPDH promotor-specific primers. f H1299 cells were transfected with or without pcDNA3.0-p65 plasmid. Nuclear proteins were extracted and subjected to EMSA assay using biotin-labeled probes containing the NF-κB binding site. The arrow indicates probes that bound to NF-κB (p65). g pGL3-enhancer vector containing PKCε promoter fragment was transfected into H1299 cells, co-transfected with control or NF-κB (p65) siRNAs and Renilla control plasmid. The relative levels of luciferase activity were normalized to the levels of untreated cells and to the levels of luciferase activity of the Renilla control plasmid. Data represent the average of three independent experiments (mean ± SD). ***P < 0.001. h pGL3-enhancer vector containing PKCε promoter fragment was transfected into H1299, co-transfected with Renilla control plasmid and pcDNA3.0 vector or pcDNA3.0-p65 plasmid. The relative levels of luciferase activity were normalized to the levels of vector control and to the levels of luciferase activity of the Renilla control plasmid. Data represent the average of three independent experiments (mean ± SD). **P < 0.01. i pGL3-enhancer vector containing PKCε promoter fragment with mutations in NF-κB binding site was transfected into H1299 cells, co-transfected with control or NF-κB (p65) siRNAs and Renilla control plasmid. The relative levels of luciferase activity were normalized to the levels of untreated cells and to the levels of luciferase activity of the Renilla control plasmid. Data represent the average of three independent experiments (mean ± SD). ns: P > 0.05. j pGL3-enhancer vector containing PKCε promoter fragment with mutations in NF-κB binding site was transfected into H1299, co-transfected with Renilla control plasmid and pcDNA3.0 vector or pcDNA3.0-p65 plasmid. The relative levels of luciferase activity were normalized to the levels of vector control and to the levels of luciferase activity of the Renilla control plasmid. Data represent the average of three independent experiments (mean ± SD). ns: P > 0.05