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. 2000 Mar;20(5):1626–1638. doi: 10.1128/mcb.20.5.1626-1638.2000

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Copyright © 2000, American Society for Microbiology

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FIG. 2 — H-Ras(V12) stimulates NF-κB-dependent transcription through PI3K- and Akt-dependent pathways. (A) Activated forms of PI3K (PI3K*) and Akt stimulate the transcriptional activity of NF-κB as effectively as H-Ras(V12). NIH 3T3 cells were cotransfected with the 3x-κB-Luc reporter (0.5 μg), an internal control plasmid reporter (pCMV-LacZ, 1 μg), and various expression constructs (1 μg each). Luciferase and β-Gal activities were assayed 24 h posttransfection, and fold luciferase activity was determined by normalizing values to total protein levels and to β-Gal enzyme levels. Results are expressed as multiples of the level of activation obtained with the mutant 3x-κB-Luc reporter, which contains mutated cis elements that are no longer capable of binding NF-κB. Data are representative of results of at least three independent experiments, and the means ± standard deviations are shown. (Gel) Immunoblot analysis demonstrates that transfected cells effectively express the various transgenes. (B) H-Ras(V12)-induced NF-κB transcriptional activity requires PI3K- and Akt-dependent signaling pathways. NIH 3T3 cells were transiently transfected with the 3x-κB-Luc reporter (0.5 μg), in either the absence (−) or presence (+) of H-Ras(V12) (1 μg). Additionally, cells were transfected with the empty vector control (VC) or with expression constructs bearing genes encoding dominant negative PI3K (ΔPI3K) or Akt (Akt T308A and Akt K179A, 1 μg each). Luciferase levels were measured 24 h posttransfection in order to avoid potential pitfalls associated with cell death. Data are multiples of the level of activation obtained with the mutant 3x-κB-Luc control, as described above. Results are representative of at least three independent experiments and were normalized to an internal β-Gal-expressing plasmid. (Gel) Immunoblot analysis shows that transfected NIH 3T3 cells express the dominant negative P13K (ΔPI3K) and Akt (DN Akt) constructs. (C) PI3K requires Akt to stimulate NF-κB-dependent transcription. NIH 3T3 cells were transfected with the 3x-κB-Luc reporter (0.5 μg), a vector control (VC), activated PI3K (PI3K*), or dominant negative Akt (Akt K179A) (1 μg each). Forty-eight h posttransfection whole-cell extracts were harvested and assayed for luciferase activity. Results are plotted as multiples of the level of activation of the vector control and are representative of three independent experiments. The means ± standard deviations are shown.

FIG. 2 — H-Ras(V12) stimulates NF-κB-dependent transcription through PI3K- and Akt-dependent pathways. (A) Activated forms of PI3K (PI3K*) and Akt stimulate the transcriptional activity of NF-κB as effectively as H-Ras(V12). NIH 3T3 cells were cotransfected with the 3x-κB-Luc reporter (0.5 μg), an internal control plasmid reporter (pCMV-LacZ, 1 μg), and various expression constructs (1 μg each). Luciferase and β-Gal activities were assayed 24 h posttransfection, and fold luciferase activity was determined by normalizing values to total protein levels and to β-Gal enzyme levels. Results are expressed as multiples of the level of activation obtained with the mutant 3x-κB-Luc reporter, which contains mutated cis elements that are no longer capable of binding NF-κB. Data are representative of results of at least three independent experiments, and the means ± standard deviations are shown. (Gel) Immunoblot analysis demonstrates that transfected cells effectively express the various transgenes. (B) H-Ras(V12)-induced NF-κB transcriptional activity requires PI3K- and Akt-dependent signaling pathways. NIH 3T3 cells were transiently transfected with the 3x-κB-Luc reporter (0.5 μg), in either the absence (−) or presence (+) of H-Ras(V12) (1 μg). Additionally, cells were transfected with the empty vector control (VC) or with expression constructs bearing genes encoding dominant negative PI3K (ΔPI3K) or Akt (Akt T308A and Akt K179A, 1 μg each). Luciferase levels were measured 24 h posttransfection in order to avoid potential pitfalls associated with cell death. Data are multiples of the level of activation obtained with the mutant 3x-κB-Luc control, as described above. Results are representative of at least three independent experiments and were normalized to an internal β-Gal-expressing plasmid. (Gel) Immunoblot analysis shows that transfected NIH 3T3 cells express the dominant negative P13K (ΔPI3K) and Akt (DN Akt) constructs. (C) PI3K requires Akt to stimulate NF-κB-dependent transcription. NIH 3T3 cells were transfected with the 3x-κB-Luc reporter (0.5 μg), a vector control (VC), activated PI3K (PI3K*), or dominant negative Akt (Akt K179A) (1 μg each). Forty-eight h posttransfection whole-cell extracts were harvested and assayed for luciferase activity. Results are plotted as multiples of the level of activation of the vector control and are representative of three independent experiments. The means ± standard deviations are shown.