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. 1999 Sep;19(9):6367–6378. doi: 10.1128/mcb.19.9.6367

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Copyright © 1999, American Society for Microbiology

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FIG. 3 — Coexpression of CBP and SRC-1/NCoA-1, GRIP-1, TIF-2, or p/CAF enhances p65-mediated transcriptional activity. (A) SRC-1/NCoA-1 potentiates NF-κB-dependent gene expression. COS-7 cells were transiently transfected with 1 μg of −578 E-selectin-CAT and 100 ng of pcDNA-p65 (lanes 2, 7, and 8) and either 3.25 μg (lanes 5, 9, and 11) or 6.5 μg (lanes 6, 10, and 12) of CMV-SRC-1/NCoA-1 and/or 3.25 μg (lanes 3, 7, and 11) or 6.5 μg (lanes 4, 8, and 12) of RSV-CBP. Forty-eight hours posttransfection, the cells were harvested and CAT activity was assayed as described in Materials and Methods. The level of activity observed upon transfection of E-selectin CAT alone was set at one. The data are presented as means; error bars, standard deviations. (B) Western blot analysis of p65 levels in transfected cell extracts. A portion of each whole-cell extract was separated by SDS–10% PAGE, transferred to nitrocellulose, and probed with a rabbit anti-p65 antibody (Rockland) as described in Materials and Methods. Following incubation with a horseradish peroxidase-conjugated donkey anti-rabbit secondary antibody, the bands were visualized by enhanced chemiluminescence (Amersham Life Science). (C) GRIP-1 and TIF-2 increase p65-dependent gene expression. COS-7 cells were transiently transfected with 1 μg of −578 E-selectin-CAT and 100 ng of pcDNA-p65 (lanes 4 to 16) and 1, 2.5, 4, or 6.6 μg of simian virus 40 (SV40)-GRIP-1, SV40-TIF-2, or RSV-CBP. Forty-eight hours posttransfection, the cells were harvested and CAT activity was assayed as described in Materials and Methods. The level of activity observed upon cotransfection of E-selectin CAT and p65 was set at one. Data are presented as means; error bars, standard deviations. (D) Western blot analysis of p65 levels in transfected cell extracts performed as described above and in Materials and Methods. (E) p/CAF potentiates p65-dependent transactivation and synergizes with CBP. COS-7 cells were transiently transfected with 1 μg of −578 E-selectin–CAT and 100 ng of pcDNA-p65 (lanes 2 and 7 to 12) and either 3.25 μg (lanes 5, 9, and 11) or 6.5 μg (lanes 6, 10, and 12) of CMV-p/CAF and/or 3.25 μg (lanes 3, 7, and 11) or 6.5 μg (lanes 4, 8, and 12) of RSV-CBP. Forty-eight hours posttransfection, the cells were harvested and CAT activity was assayed as described in Materials and Methods. The level of activity observed upon transfection of E-selectin CAT alone was set at one. Data are presented as means; error bars, standard deviations. (F) Western blot analysis of p65 levels in cell extracts performed as described above and in Materials and Methods.

FIG. 3 — Coexpression of CBP and SRC-1/NCoA-1, GRIP-1, TIF-2, or p/CAF enhances p65-mediated transcriptional activity. (A) SRC-1/NCoA-1 potentiates NF-κB-dependent gene expression. COS-7 cells were transiently transfected with 1 μg of −578 E-selectin-CAT and 100 ng of pcDNA-p65 (lanes 2, 7, and 8) and either 3.25 μg (lanes 5, 9, and 11) or 6.5 μg (lanes 6, 10, and 12) of CMV-SRC-1/NCoA-1 and/or 3.25 μg (lanes 3, 7, and 11) or 6.5 μg (lanes 4, 8, and 12) of RSV-CBP. Forty-eight hours posttransfection, the cells were harvested and CAT activity was assayed as described in Materials and Methods. The level of activity observed upon transfection of E-selectin CAT alone was set at one. The data are presented as means; error bars, standard deviations. (B) Western blot analysis of p65 levels in transfected cell extracts. A portion of each whole-cell extract was separated by SDS–10% PAGE, transferred to nitrocellulose, and probed with a rabbit anti-p65 antibody (Rockland) as described in Materials and Methods. Following incubation with a horseradish peroxidase-conjugated donkey anti-rabbit secondary antibody, the bands were visualized by enhanced chemiluminescence (Amersham Life Science). (C) GRIP-1 and TIF-2 increase p65-dependent gene expression. COS-7 cells were transiently transfected with 1 μg of −578 E-selectin-CAT and 100 ng of pcDNA-p65 (lanes 4 to 16) and 1, 2.5, 4, or 6.6 μg of simian virus 40 (SV40)-GRIP-1, SV40-TIF-2, or RSV-CBP. Forty-eight hours posttransfection, the cells were harvested and CAT activity was assayed as described in Materials and Methods. The level of activity observed upon cotransfection of E-selectin CAT and p65 was set at one. Data are presented as means; error bars, standard deviations. (D) Western blot analysis of p65 levels in transfected cell extracts performed as described above and in Materials and Methods. (E) p/CAF potentiates p65-dependent transactivation and synergizes with CBP. COS-7 cells were transiently transfected with 1 μg of −578 E-selectin–CAT and 100 ng of pcDNA-p65 (lanes 2 and 7 to 12) and either 3.25 μg (lanes 5, 9, and 11) or 6.5 μg (lanes 6, 10, and 12) of CMV-p/CAF and/or 3.25 μg (lanes 3, 7, and 11) or 6.5 μg (lanes 4, 8, and 12) of RSV-CBP. Forty-eight hours posttransfection, the cells were harvested and CAT activity was assayed as described in Materials and Methods. The level of activity observed upon transfection of E-selectin CAT alone was set at one. Data are presented as means; error bars, standard deviations. (F) Western blot analysis of p65 levels in cell extracts performed as described above and in Materials and Methods.