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Results: 6

1.
Figure 5

Figure 5. From: Induction of Prostatic Intraepithelial Neoplasia and Modulation of Androgen Receptor by ETS Variant 1/ETS-Related Protein 81.

Mapping of interaction domains. A, Flag-tagged fragments of AR were coexpressed with Myc-tagged ETV1. After anti-Flag immunoprecipitation, coprecipitated ETV1 was revealed by anti-Myc Western blotting. A/B, activation domain; C, DNA binding domain; D, hinge region; E/F, ligand binding domain of AR. B, coprecipitation of AR with Myc-tagged fragments of ETV1. Sketch of ETV1 at the top shows its activation domains and DNA binding ETS domain.

Sook Shin, et al. Cancer Res. 2009 October 15;69(20):8102-8110.
2.
Figure 4

Figure 4. From: Induction of Prostatic Intraepithelial Neoplasia and Modulation of Androgen Receptor by ETS Variant 1/ETS-Related Protein 81.

Interaction of ETV1 and ERG with AR. A, binding of Flag-tagged AR to Myc-tagged ETV1 was assessed by coimmunoprecipitation in the presence and absence of mibolerone. B, mibolerone-treated LNCaP cells were lysed and ETV1 was immunoprecipitated. Coprecipitated AR was revealed by anti-AR Western blotting. C, GST pull-down experiments in the presence or absence of 1 nmol/L mibolerone. Indicated GST fusion proteins or GST was used to pull down Flag-tagged, baculovirus-expressed AR. Bottom, amounts of GST fusion proteins used by Coomassie staining. D, coimmunoprecipitation of AR with Myc-tagged ERG in the presence of mibolerone.

Sook Shin, et al. Cancer Res. 2009 October 15;69(20):8102-8110.
3.
Figure 6

Figure 6. From: Induction of Prostatic Intraepithelial Neoplasia and Modulation of Androgen Receptor by ETS Variant 1/ETS-Related Protein 81.

A, activation of a luciferase gene driven by the ETS site E74. Where indicated, AR and/or ETV1 were transfected into RK13 cells and stimulated with 0.5 nmol/L mibolerone. B, analogous stimulation of an AR binding site–driven luciferase reporter in the presence or absence of 1 nmol/L mibolerone. Western blotting (inset) shows that AR levels were unaffected by ETV1 expression in the presence of mibolerone. C, as in B with ERG instead of ETV1.

Sook Shin, et al. Cancer Res. 2009 October 15;69(20):8102-8110.
4.
Figure 3

Figure 3. From: Induction of Prostatic Intraepithelial Neoplasia and Modulation of Androgen Receptor by ETS Variant 1/ETS-Related Protein 81.

Binding of ETV1 to the PSA enhancer. A, activation of the PSA enhancer by ETV1. Left, scheme of the PSA enhancer and two truncations thereof; right, luciferase reporter assays with these enhancer fragments in LNCaP cells transfected with or without ETV1 (250 ng). Where indicated, cells were stimulated with 1 nmol/L mibolerone. B, binding of ETV1 to the 10 ETS sites in the PSA enhancer was assessed in electrophoretic mobility shift assays. Where indicated, anti-ETV1 antibody (Ab) was added to the binding reaction. C, comparison of the activities of the wild-type and mETS#8 (mutation of ETS site 8) −4324/−3875 PSA enhancer in LNCaP cells. Where indicated, ETV1 (600 ng) was cotransfected and 0.25 nmol/L mibolerone was used.

Sook Shin, et al. Cancer Res. 2009 October 15;69(20):8102-8110.
5.
Figure 1

Figure 1. From: Induction of Prostatic Intraepithelial Neoplasia and Modulation of Androgen Receptor by ETS Variant 1/ETS-Related Protein 81.

ETV1 and prostatic lesions. A, ETV1 mRNA expression derived by microarray from normal prostates (n = 50), PINs (n = 5), Gleason pattern 3 (GP3; n = 32), Gleason pattern 4 (GP4; n = 46), Gleason pattern 5 (GP5; n = 22), and metastases (n = 7). B, ETV1 and ERG expression derived by RT-PCR from high-risk prostate cancer patients. Relapse indicates patients that developed systemic progression within 5 y after prostatectomy, whereas controls remained free of disease for at least 8 y. Dashed line, boundary of significant ETV1 or ERG overexpression. C, H&E-stained sections of the anterior (top) and ventral (bottom) prostate of transgenic ETV1 mouse 650. Right, larger magnification of PIN areas. D, similar for a 1-y-old control mouse.

Sook Shin, et al. Cancer Res. 2009 October 15;69(20):8102-8110.
6.
Figure 2

Figure 2. From: Induction of Prostatic Intraepithelial Neoplasia and Modulation of Androgen Receptor by ETS Variant 1/ETS-Related Protein 81.

Activation of the PSA gene by ETV1. A, ETV1 or its 6xA mutant (300 ng) was cotransfected with the 5.8-kb PSA luciferase reporter construct into LNCaP cells stimulated with or without 0.1 nmol/L mibolerone. B, anti-Myc Western blot showing comparable expression of Myc-tagged wild-type ETV1 and its 6xA mutant in LNCaP cells. C, ETV1 down-regulation by three different shRNAs inhibits expression of endogenous PSA mRNA and protein in LNCaP cells. Top two rows, agarose gels revealing reverse-transcribed mRNA; bottom three rows, Western blots. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA and actin protein levels were determined as loading controls. D, top, scheme of the PSA enhancer and proximal promoter with its AR binding sites in Roman numerals and potential ETS sites in Arabic numerals. AR site III is the most important AR binding site. Numbering is according to ref. 31. Bottom, chromatin immunoprecipitation assays assessing the binding of ETV1 and AR to the PSA enhancer, proximal promoter, and 3′-UTR in LNCaP cells before and after 1 or 8 h of stimulation with 1 nmol/L mibolerone.

Sook Shin, et al. Cancer Res. 2009 October 15;69(20):8102-8110.

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