Journal:

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

doi:

Figure Lengend Snippet: AIB1 is phosphorylated in vivo. (A) In vitro translated (IVT) AIB1 was labeled with [35S]Met and immunoprecipitated (IP) with either preimmune serum (lane 2) or anti-AIB1 antibodies (lane 3). Subconfluent MCF-7 cells were labeled with [32P]orthophosphate for 6 h and immunoprecipitated with either preimmune serum (lane 4) or anti-AIB1 antibodies (lane 5). Immune complexes were resolved by SDS–8% PAGE. The gel was dried and exposed for autoradiography. The figure is representative of four independent experiments. (B) The band corresponding to AIB1 was excised from the gel and treated with HCl to total hydrolysis, analyzed by TLC together with phosphoamino acid markers, developed with ninhydrin to reveal the mobility of nonradioactive phosphoaminoacids (indicated by dashed circles), and subsequently exposed for autoradiography. (C) Breast cancer cell lines MCF-7, BT-474, and MDA-MB-468 were analyzed by Western blotting (WB) for the expression of AIB1. Each well was loaded with 30 μg of total protein from whole-cell lysates (upper panel). In parallel, cells were labeled with [32P]orthophosphate for 6 h, and 0.8 mg of total protein from each cell line lysate was immunoprecipitated with anti-AIB1 antibodies (lower panel).

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: In Vivo, In Vitro, Labeling, Immunoprecipitation, Autoradiography, Western Blot, Expressing

Journal:

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

doi:

Figure Lengend Snippet: AIB1 is a substrate of MAPK. Proliferating MCF-7 cells were lysed, and aliquots containing 3 mg of total protein were used for immunoprecipitation (IP) and kinase reactions. Lysates were immunoprecipitated with preimmune serum (lane 1) or with immunopurified anti-AIB1 polyclonal antibodies (lanes 3 to 5). AIB1 immune complexes were split into thirds. One third received no further treatment (lane 3). The other two thirds were treated with protein λ-phosphatase (λPPase) (lane 4), and after a washing, one third was further resuspended in 30 μl of buffer containing 100 μM ATP and treated with 0.1 μg of active Erk2 (Upstate Biotechnology) for 1 hour at 30°C (lane 5). In parallel, the other sample was treated with 0.1 μCi of [32P]ATP and resolved independently. Gels were either Western immunoblotted (WB) with AIB1 antibodies (upper panel) or dried on Whatman paper and exposed for autoradiography (lower panel). Similar results were obtained in three 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: Immunoprecipitation, Western Blot, Autoradiography

Journal:

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

doi:

Figure Lengend Snippet: 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 (RΔF). (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 (RΔF) 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, Plasmid Preparation

Journal:

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

doi:

Figure Lengend Snippet: 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 (−) or vector containing activated MEK1 (RΔF). (A) After 48 h, endogenous Erk2 was immunoprecipitated to determine MAPK activity, using MBP as a substrate. (B) In parallel, 25 μg 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

Journal:

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

doi:

Figure Lengend Snippet: Regulation of steroid receptor transcriptional complexes by MAPK activation. Growth factors such as EGF and IGF-1 stimulate the Ras-MAPK cascade via activation of their respective tyrosine kinase receptors. Activated MAPK may then phosphorylate nuclear targets including ER and AIB1 to modulate gene transcription in response to growth factor and/or steroid stimulation.

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