Journal:

Article Title: Transcriptional Activation by NF-?B Requires Multiple Coactivators

doi:

Figure Lengend Snippet: Coactivator requirements for NF-κB-dependent gene expression. Plasmids consisting of a LacZ reporter under the transcriptional control of the E-selectin promoter were injected into the nuclei of Rat-1 cells in the presence of either preimmune IgG or affinity-purified antibodies to the indicated coactivators. The expression of the reporter plasmid was monitored by staining with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) and quantitated based on the percentage of injected cells that stained blue. (A) Effects of nuclear microinjection of anti-CBP, SRC-1/NCoA-1, and pCAF antibodies on p65-induced E-selectin-lacZ reporter gene expression. Photomicrographs of rhodamine-stained injected cells (top panel) and the corresponding phase-contrast pictures (lower panel) display typical results. (B) Coactivator requirements of NF-κB-dependent gene expression. Results were repeated in three separate experiments with more than 200 cells injected for each data point; data are expressed as means, and error bars represent standard errors of the means. (C) Effect of nuclear microinjection of anti-CBP, SRC-1/NCoA-1, and p/CAF antibodies on Sp1-LacZ and CMV-LacZ reporter constructs.

Article Snippet: The samples were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) (10% polyacrylamide) followed by Western blotting with the following antibodies: rabbit anti-CBP (Santa Cruz Biotechnology Inc.), rabbit anti-p/CAF (Yoshihiro Nakatani), mouse anti-SRC-1/NCoA-1 (Myles Brown), and rabbit anti-c-JUN (Santa Cruz Biotechnology Inc.).

Techniques: Expressing, Injection, Affinity Purification, Plasmid Preparation, Staining, Construct

Journal:

Article Title: Transcriptional Activation by NF-?B Requires Multiple Coactivators

doi:

Figure Lengend Snippet: 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.

Article Snippet: The samples were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) (10% polyacrylamide) followed by Western blotting with the following antibodies: rabbit anti-CBP (Santa Cruz Biotechnology Inc.), rabbit anti-p/CAF (Yoshihiro Nakatani), mouse anti-SRC-1/NCoA-1 (Myles Brown), and rabbit anti-c-JUN (Santa Cruz Biotechnology Inc.).

Techniques: Activity Assay, Expressing, Transfection, Western Blot, Incubation, Cotransfection

Journal:

Article Title: Transcriptional Activation by NF-?B Requires Multiple Coactivators

doi:

Figure Lengend Snippet: NF-κB-dependent transactivation in vitro requires specific functional domains of SRC-1/NCoA-1. (A) Schematic representation of SRC-1/NCoA-1 expression plasmids used in the overexpression experiments. (B) The N terminus of SRC-1/NCoA-1 is required for potentiation of p65-dependent transactivation. COS-7 cells were transiently transfected with 1 μg of −578 E-selectin-CAT, 100 ng of pcDNA-p65, and 0.5, 1, 4, or 8 μg of either wild-type CMV-SRC-1/NCoA-1 (WT), CMV-SRC-1-NR/CBP, or CMV-SRC-1 ΔN. 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.

Article Snippet: The samples were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) (10% polyacrylamide) followed by Western blotting with the following antibodies: rabbit anti-CBP (Santa Cruz Biotechnology Inc.), rabbit anti-p/CAF (Yoshihiro Nakatani), mouse anti-SRC-1/NCoA-1 (Myles Brown), and rabbit anti-c-JUN (Santa Cruz Biotechnology Inc.).

Techniques: In Vitro, Functional Assay, Expressing, Over Expression, Transfection, Activity Assay, Cotransfection

Journal:

Article Title: Transcriptional Activation by NF-?B Requires Multiple Coactivators

doi:

Figure Lengend Snippet: Coactivator LXXLL motif specificity in NF-κB-dependent gene expression. (A) The LXD2 and LXD4 domains of SRC-1/NCoA-1 are required for NF-κB-mediated transcription. Plasmids consisting of a LacZ reporter under the transcriptional control of the E-selectin promoter were injected into the nuclei of Rat-1 cells in the presence of either preimmune IgG or an affinity-purified antibody to SRC-1/NCoA-1. The expression of the reporter plasmid was monitored by X-Gal staining and quantitated based on the percentage of injected cells that stained blue. Rescue experiments were performed by coinjecting the indicated expression plasmids. (B) Coinjection of NCoA-2 expression plasmid does not rescue the inhibitory effect of the anti-SRC-1/NCoA-1 antibody on p65-dependent transcription. (C) Coinjection of NCoA-2 expression plasmid rescues the inhibitory effect of the anti-SRC-1/NCoA-1 antibody on RAR-dependent transcription. In all panels, data are means from three separate experiments; error bars represent standard errors of the means.

Article Snippet: The samples were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) (10% polyacrylamide) followed by Western blotting with the following antibodies: rabbit anti-CBP (Santa Cruz Biotechnology Inc.), rabbit anti-p/CAF (Yoshihiro Nakatani), mouse anti-SRC-1/NCoA-1 (Myles Brown), and rabbit anti-c-JUN (Santa Cruz Biotechnology Inc.).

Techniques: Expressing, Injection, Affinity Purification, Plasmid Preparation, Staining

Journal:

Article Title: Transcriptional Activation by NF-?B Requires Multiple Coactivators

doi:

Figure Lengend Snippet: CBP/p300, SRC-1/NCoA-1, and p/CAF are recruited to a DNA-bound p65-p50 heterodimer. (Left panel) Coactivators are recruited to a DNA-bound p65-p50 heterodimer. A biotinylated oligonucleotide containing two NF-κB binding sites was bound to streptavidin-paramagnetic beads and loaded with 0 μg (lane 2), 1 μg (lane 3), or 3 μg of His-p50–His-p65 (lane 4). After a wash, the DNA-bound heterodimers were incubated with 2 mg of K562 nuclear extracts and then washed, and bound proteins were identified by Western blotting as described in Materials and Methods. Lane 1 represents 200 μg, or 1/10 of the total K562 input. The antibodies used for Western blotting are indicated on the left. (Right panel) The p50 homodimer inhibits recruitment of CBP/p300, SRC-1/NCoA-1, and p/CAF. A biotinylated oligonucleotide containing two NF-κB binding sites was bound to streptavidin-paramagnetic beads and loaded with 1 or 3 μg of either p50-p65 (lanes 6 and 7) or the p50–p50 homodimer (lanes 8 and 9). After a wash, the DNA bound dimers were incubated with 2 mg of K562 nuclear extracts and then washed, and bound proteins were identified by Western blot analysis, as described in Materials and Methods. Lane 5 (Input) represents 200 μg, or 1/10, of the total K562 input.

Article Snippet: The samples were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) (10% polyacrylamide) followed by Western blotting with the following antibodies: rabbit anti-CBP (Santa Cruz Biotechnology Inc.), rabbit anti-p/CAF (Yoshihiro Nakatani), mouse anti-SRC-1/NCoA-1 (Myles Brown), and rabbit anti-c-JUN (Santa Cruz Biotechnology Inc.).

Techniques: Binding Assay, Incubation, Western Blot