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Androgen Receptor and PIN1 in Prostate Cancer

von Dott. Raffaele La Montagna

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Statistik und Sichtungsnachweis dieser Seite findet sich am Artikelende
[1.] Rlm/Fragment 056 01 - Diskussion
Zuletzt bearbeitet: 2014-12-05 19:06:54 Singulus
Fragment, Gesichtet, Rizzolio 2010, Rlm, SMWFragment, Schutzlevel sysop, Verschleierung

Typus
Verschleierung
Bearbeiter
Graf Isolan
Gesichtet
Yes.png
Untersuchte Arbeit:
Seite: 56, Zeilen: 1-15
Quelle: Rizzolio 2010
Seite(n): 65-66, Zeilen: 65:11-17.(17-19).20-22 - 66:1-5
The remaining bacteria were then resuspended in NENT buffer plus 2% of N-lauryl-sarcosine, then pelleted and finally, the supernatants were collected again. The combined supernatants were incubated with glutathione agarose beads (Sigma Inc., St Louis, MO, USA) overnight at 4 oC. The agarose was then pelleted and washed three times in NENT buffer. The GST protein was analyzed by electrophoresis gel and blue coomassie staining. 1mg of protein was pulled-down with 10 ug of GST or GST-Pin1.

Co-immunoprecipitation assay

Sub-confluent LNcaP cells were harvested and proteins were prepared as follows: the cell pellet was resuspended in lysis buffer (20 mM Tris HCl pH 8, 137 mM NaCl, 10% glycerol, 1% NP40, 2 mM EDTA). 1mg of proteins was immunoprecipitated, utilizing 4 μg of PIN1, AR antibody or mouse IgG overnight at 4°C Extracts were incubated with antibodies and protein A/G beads (Pierce) for 3 h at 4°C.

[Page 65]

The remaining bacteria were then resuspended in NENT buffer plus 2% of N-lauryl-sarcosine, then pelleted and finally, the supernatants were collected again. The combined supernatants were incubated with glutathione agarose beads (Sigma Inc., St Louis, MO, USA) overnight at 4 oC. The agarose was then pelleted and washed three times in NENT buffer. The GST protein was analyzed by electrophoresis gel and blue coomassie staining. 1mg of protein was pulled-down with 10 ug of GST or GST-PIN1. To dephosphorylate proteins, 1mg of protein lysate was treated with 50 U of shrimp alkaline phosphatase for 1h at 37 oC.

Co-immunoprecipitation assay

Sub-confluent T98G cells were harvested and nuclear/cytoplasmatic proteins were prepared as follows: the cell pellet was resuspended in NP40 lysis

[Page 66]

buffer (0.01M Tris-HCl, 0.01M NaCl, 0.003M MgCl2, 0.03M Sucrose, 0.5% NP40) to prepare the cytoplasmatic fraction. Afterwards, nuclei were pelleted and resuspended in lysis buffer (20 mM Tris HCl pH 8, 137 mM NaCl, 10% glycerol, 1% NP40, 2 mM EDTA). 1mg of proteins was immunoprecipitated, utilizing 4 μg of PIN1 antibody.

Anmerkungen

Although nearly identical, nothing has been marked as a citation. Continued from previous page.

Sichter
(Graf Isolan), SleepyHollow02


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