pegfp-foxa1-n3  (SignaGen)

Journal: The Prostate

Article Title: FOXA1 modulates EAF2 regulation of AR transcriptional activity, cell proliferation and migration in prostate cancer cells

doi: 10.1002/pros.22982

Figure Lengend Snippet: (A) 2.5 μg FLAG-EAF2 and 2.5 μg GFP-FOXA1 were transfected into HEK 293 cells separately or in combination, and then whole cell lysates were prepared for co-immunoprecipitation with anti-GFP or anti-FLAG antibody-conjugated agarose beads. FLAG-EAF2 and GFP-FOXA1 could be pulled down by each other. (B) Co-immunoprecipitation of endogenous EAF2 and FOXA1 was performed using nuclear extracts prepared from LNCaP cells with rabbit anti-FOXA1 antibody or control rabbit IgG. Co-precipitated EAF2 was detected by Western blot using a monoclonal mouse anti-EAF2 antibody. (C) 2.5 μg Myc-tagged full-length EAF2, EAF2 deletion mutants 5 μg Myc-EAF2 (amino acids (aa) 1-113), 5 μg Myc-EAF2 (aa 1-161), or 2.5 μg Myc-EAF2 (aa 113-260) were co-transfected with 2.5 μg GFP-FOXA1 into HEK 293 cells. Co-immunoprecipitation was performed with anti-Myc antibody and GFP-FOXA1 was detected with anti-GFP antibody.

Article Snippet: When the cells reached 80% confluence, cells were transfected with 0.83 μg pEGFP-N3, 4.17 μg CMV-HA, 4.17 μg CMV-HA-EAF2 and/or 0.83 μg pEGFP-FOXA1-N3 as indicated using PolyJet In Vitro Transfection reagent (SignaGen, Gaithersburg, MD, USA).

Techniques: Transfection, Immunoprecipitation, Western Blot

Journal: The Prostate

Article Title: FOXA1 modulates EAF2 regulation of AR transcriptional activity, cell proliferation and migration in prostate cancer cells

doi: 10.1002/pros.22982

Figure Lengend Snippet: (A) Two different siRNAs targeting EAF2 were used separately at 100 pmol each to knockdown EAF2 expression in LNCaP cells. Endogenous protein levels of FOXA1, AR and EAF2 were detected by Western blot using anti-FOXA1 anti-AR, and anti-EAF2 antibodies. The protein level of FOXA1 increased upon EAF2 knockdown. (B) Total RNA was isolated from LNCaP cells after knockdown of EAF2. Relative mRNA levels of FOXA1 normalized against GAPDH were determined using real-time PCR. (C) HEK293 cells were co-transfected with 0.2 μg FOXA1 and increasing amounts of GFP-EAF2. Expression levels of FOXA1and EAF2 were determined by Western blot. GAPDH protein was detected as a loading control.

Article Snippet: When the cells reached 80% confluence, cells were transfected with 0.83 μg pEGFP-N3, 4.17 μg CMV-HA, 4.17 μg CMV-HA-EAF2 and/or 0.83 μg pEGFP-FOXA1-N3 as indicated using PolyJet In Vitro Transfection reagent (SignaGen, Gaithersburg, MD, USA).

Techniques: Expressing, Western Blot, Isolation, Real-time Polymerase Chain Reaction, Transfection

Journal: The Prostate

Article Title: FOXA1 modulates EAF2 regulation of AR transcriptional activity, cell proliferation and migration in prostate cancer cells

doi: 10.1002/pros.22982

Figure Lengend Snippet: LNCaP cells were cultured in the presence of indicated siRNAs for 24 hours and then treated with 1 nM R1881 for 48 h. Subsequently, total RNA was extracted and mRNA levels of EAF2 (A), FOXA1 (B), AR (C), PSA (D) and TMPRSS2 (E) were determined by real-time PCR. (F) LNCaP cells were transfected with siRNA targeting the indicated proteins followed by transfection with 0.5 μg PSA-promoter driven fire-fly luciferase vector and 0.05 μg pCMV-Renilla in the presence or absence of the androgen analog R1881 (1 nM). Luciferase assays were performed as described in the Materials and Methods.

Article Snippet: When the cells reached 80% confluence, cells were transfected with 0.83 μg pEGFP-N3, 4.17 μg CMV-HA, 4.17 μg CMV-HA-EAF2 and/or 0.83 μg pEGFP-FOXA1-N3 as indicated using PolyJet In Vitro Transfection reagent (SignaGen, Gaithersburg, MD, USA).

Techniques: Cell Culture, Real-time Polymerase Chain Reaction, Transfection, Luciferase, Plasmid Preparation

Journal: The Prostate

Article Title: FOXA1 modulates EAF2 regulation of AR transcriptional activity, cell proliferation and migration in prostate cancer cells

doi: 10.1002/pros.22982

Figure Lengend Snippet: Effect of EAF2 and/or FOXA1 transfection on LNCaP cell colony formation. Left panel shows the colonies formed by LNCaP cells transfected with indicated expression vectors. Right panel shows the quantitative analysis of the colonies of the left panel. Transfection and quantitative analysis of colonies were conducted as described in Materials and Methods.

Article Snippet: When the cells reached 80% confluence, cells were transfected with 0.83 μg pEGFP-N3, 4.17 μg CMV-HA, 4.17 μg CMV-HA-EAF2 and/or 0.83 μg pEGFP-FOXA1-N3 as indicated using PolyJet In Vitro Transfection reagent (SignaGen, Gaithersburg, MD, USA).

Techniques: Transfection, Expressing