Figure 5.
Figure 5. Overexpression of Bcl-3 inhibits LPS-induced transactivation of the TNF-α promoter. (A) RAW cells were transiently cotransfected with the TNF-α promoter-luciferase construct (10 ng) and the expression vectors for full-length Bcl-3 or ΔN (10 ng, 100 ng, or 500 ng), as indicated. After 24 hours of transfection, cells were treated with or without 1 μg/mL LPS for 8 hours and then the luciferase activities were measured. Data are representative of 3 independent experiments yielding similar results. Data are expressed as relative fold activation compared with the nonstimulated (-) set. (B) RAW cells were transiently cotransfected with the IL-6 promoter-luciferase construct (100 ng) and the expression vector for Bcl-3 (10 ng, 100 ng, or 500 ng), as indicated. The cells were treated with 1 μg/mL LPS for 8 hours and luciferase activity was detected. Representative results of 3 independent experiments are shown. (C) The 293 cells were transfected with various combinations of pEF-BOS-Myc-Bcl-3, pcDNA3.1-Flag-p50, and pcDNA3.1-Flag-p52 in a 6-well dish (each 1 μg/well). Total amount of transfected DNA was kept at 2 μg by adding pEF-BOS or pcDNA 3.1. p50 and p52 were immunoblotted by anti-Flag antibody (M2 monoclonal antibody). Bcl-3 was immunoblotted by rabbit polyclonal anti-c-Myc antibody. Plasmids transfected are indicated on top of the panel. (D) RAW cells expressing Myc-Bcl-3 and Flag-p50 were stimulated with 100 ng/mL LPS for 2 hours, and chromatin immunoprecipitation assays were performed with α-Myc or α-Flag antibodies. The detection of the immunoprecipitated TNF-α promoter (left panel) or IL-6 promoter (right panel) was analyzed by PCR with promoter-specific primers. Representative of 3 independent experiments. (E) RAW cells were transiently cotransfected with pELAM-1, Flag-p50 (0, 1, 10, 100 ng/well), and pEF-BOS Bcl-3 (100 ng/well) expression plasmids. After 24 hours of transfection, cells were treated with 1 μg/mL LPS for 8 hours and then luciferase activity was detected. Data are expressed as relative fold activation compared with the nontransfected set. Error bars indicate SD.

Overexpression of Bcl-3 inhibits LPS-induced transactivation of the TNF-α promoter. (A) RAW cells were transiently cotransfected with the TNF-α promoter-luciferase construct (10 ng) and the expression vectors for full-length Bcl-3 or ΔN (10 ng, 100 ng, or 500 ng), as indicated. After 24 hours of transfection, cells were treated with or without 1 μg/mL LPS for 8 hours and then the luciferase activities were measured. Data are representative of 3 independent experiments yielding similar results. Data are expressed as relative fold activation compared with the nonstimulated (-) set. (B) RAW cells were transiently cotransfected with the IL-6 promoter-luciferase construct (100 ng) and the expression vector for Bcl-3 (10 ng, 100 ng, or 500 ng), as indicated. The cells were treated with 1 μg/mL LPS for 8 hours and luciferase activity was detected. Representative results of 3 independent experiments are shown. (C) The 293 cells were transfected with various combinations of pEF-BOS-Myc-Bcl-3, pcDNA3.1-Flag-p50, and pcDNA3.1-Flag-p52 in a 6-well dish (each 1 μg/well). Total amount of transfected DNA was kept at 2 μg by adding pEF-BOS or pcDNA 3.1. p50 and p52 were immunoblotted by anti-Flag antibody (M2 monoclonal antibody). Bcl-3 was immunoblotted by rabbit polyclonal anti-c-Myc antibody. Plasmids transfected are indicated on top of the panel. (D) RAW cells expressing Myc-Bcl-3 and Flag-p50 were stimulated with 100 ng/mL LPS for 2 hours, and chromatin immunoprecipitation assays were performed with α-Myc or α-Flag antibodies. The detection of the immunoprecipitated TNF-α promoter (left panel) or IL-6 promoter (right panel) was analyzed by PCR with promoter-specific primers. Representative of 3 independent experiments. (E) RAW cells were transiently cotransfected with pELAM-1, Flag-p50 (0, 1, 10, 100 ng/well), and pEF-BOS Bcl-3 (100 ng/well) expression plasmids. After 24 hours of transfection, cells were treated with 1 μg/mL LPS for 8 hours and then luciferase activity was detected. Data are expressed as relative fold activation compared with the nontransfected set. Error bars indicate SD.

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