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FIG. 4. MAPK stimulates AIB1 transcriptional activity. (A) COS cells were transiently transfected with <t>Gal4-DBD</t> fused to full-length or deletion mutants of AIB1, together with a luciferase reporter containing five copies of UAS. tk-lacZ was used as an internal control of the transfection. Transfections were performed in the presence of vector alone, MKP1, or activated MEK1 (RDF). (B) Structural features of full-length AIB1 and the two deletion mutants. Values represent the ratio between the fold induction observed in the presence of a permanently activated MAPK (RDF) and that observed in the presence of the phosphatase MKP1, as an indication of sensitivity to regulation by MAPK. Striped boxes indicate the two transactivation domains, AD1 and AD2; LXXLL, NR-interacting domains; Poly Q, stretch of polyglutamines.
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FIG. 4. MAPK stimulates AIB1 transcriptional activity. (A) COS cells were transiently transfected with Gal4-DBD fused to full-length or deletion mutants of AIB1, together with a luciferase reporter containing five copies of UAS. tk-lacZ was used as an internal control of the transfection. Transfections were performed in the presence of vector alone, MKP1, or activated MEK1 (RDF). (B) Structural features of full-length AIB1 and the two deletion mutants. Values represent the ratio between the fold induction observed in the presence of a permanently activated MAPK (RDF) and that observed in the presence of the phosphatase MKP1, as an indication of sensitivity to regulation by MAPK. Striped boxes indicate the two transactivation domains, AD1 and AD2; LXXLL, NR-interacting domains; Poly Q, stretch of polyglutamines.

Journal: Molecular and Cellular Biology

Article Title: AIB1 Is a Conduit for Kinase-Mediated Growth Factor Signaling to the Estrogen Receptor

doi: 10.1128/mcb.20.14.5041-5047.2000

Figure Lengend Snippet: FIG. 4. MAPK stimulates AIB1 transcriptional activity. (A) COS cells were transiently transfected with Gal4-DBD fused to full-length or deletion mutants of AIB1, together with a luciferase reporter containing five copies of UAS. tk-lacZ was used as an internal control of the transfection. Transfections were performed in the presence of vector alone, MKP1, or activated MEK1 (RDF). (B) Structural features of full-length AIB1 and the two deletion mutants. Values represent the ratio between the fold induction observed in the presence of a permanently activated MAPK (RDF) and that observed in the presence of the phosphatase MKP1, as an indication of sensitivity to regulation by MAPK. Striped boxes indicate the two transactivation domains, AD1 and AD2; LXXLL, NR-interacting domains; Poly Q, stretch of polyglutamines.

Article Snippet: Polyclonal antibodies against Gal4-DBD were purchased from Santa Cruz Biotechnology (no. sc-577) and used to immunoprecipitate Gal4-DBD–AIB1 proteins from transient transfected cells.

Techniques: Activity Assay, Transfection, Luciferase, Control, Plasmid Preparation

FIG. 5. Activation of MAPK recruits p300 to AIB1 complexes. Gal4-DBD alone or fused to AIB1 truncation mutants (amino acids 556 to 1420 or 578 to 1131) was transfected into BOSC cells along with pCIp300. The transfections also included vector alone (2) or vector containing activated MEK1 (RDF). (A) After 48 h, endogenous Erk2 was immunoprecipitated to determine MAPK activity, using MBP as a substrate. (B) In parallel, 25 mg of whole-cell extract was analyzed for expression of p300 by Western blotting blot (WB). Lysates were further immunoprecipitated with a polyclonal antibody to Gal4-DBD. (C and D) Immune complexes were probed for p300 (C) and Gal4-DBD proteins (D). (E) In parallel, immune complexes were analyzed for HAT activity. Similar results were obtained in five independent experiments.

Journal: Molecular and Cellular Biology

Article Title: AIB1 Is a Conduit for Kinase-Mediated Growth Factor Signaling to the Estrogen Receptor

doi: 10.1128/mcb.20.14.5041-5047.2000

Figure Lengend Snippet: FIG. 5. Activation of MAPK recruits p300 to AIB1 complexes. Gal4-DBD alone or fused to AIB1 truncation mutants (amino acids 556 to 1420 or 578 to 1131) was transfected into BOSC cells along with pCIp300. The transfections also included vector alone (2) or vector containing activated MEK1 (RDF). (A) After 48 h, endogenous Erk2 was immunoprecipitated to determine MAPK activity, using MBP as a substrate. (B) In parallel, 25 mg of whole-cell extract was analyzed for expression of p300 by Western blotting blot (WB). Lysates were further immunoprecipitated with a polyclonal antibody to Gal4-DBD. (C and D) Immune complexes were probed for p300 (C) and Gal4-DBD proteins (D). (E) In parallel, immune complexes were analyzed for HAT activity. Similar results were obtained in five independent experiments.

Article Snippet: Polyclonal antibodies against Gal4-DBD were purchased from Santa Cruz Biotechnology (no. sc-577) and used to immunoprecipitate Gal4-DBD–AIB1 proteins from transient transfected cells.

Techniques: Activation Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Activity Assay, Expressing, Western Blot