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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 055 01 - Diskussion
Zuletzt bearbeitet: 2014-12-05 19:04:14 Singulus
Fragment, Gesichtet, Rizzolio 2010, Rlm, SMWFragment, Schutzlevel sysop, Verschleierung

Typus
Verschleierung
Bearbeiter
Graf Isolan
Gesichtet
Yes.png
Untersuchte Arbeit:
Seite: 55, Zeilen: 1-2, 4-8, 10-18
Quelle: Rizzolio 2010
Seite(n): 60-61, 65, Zeilen: 60:14-18.19-21 - 61:1-2; 65:3-11
[Briefly, 1x106 293FT cells (Invitrogen Corp, Carlsbad, CA, USA) were transfected with 2.25 μg of PAX2 packaging plasmid, 0.75 μg of PMD2G envelop plasmid and 3μg of pLKO.1 hairpin vector utilizing] 30 μl of Fugene HD (Roche Applied Science, Indianapolis, IN, USA) in 10 cm plate. Polyclonal populations of Pin1 kd and scrambled cells were generated by infection with 1 MOI (multiplicity of infectious) of shRNA lentiviral particles. 2.5x105 cells were plated in a multi 6 wells plate. The day after, the cells were transduced with 1 MOI of lentiviral particle in 10% FBS MEM medium. After 3 days post-infection, the cells were selected with 2 μg/ml of puromycin (Sigma-Aldrich, St Louis, MO, USA) for 1 week [sic]

Pull-down analyses

GST and GST-Pin1 proteins were produced in BL21 bacteria cells. Cells were grown to mid log phase and then induced to express protein by adding 0.25mM of isopropyl-1-thio-b-D-galactopyranoside (IPTG, Roche Applied Science, Indianapolis, IN, USA). The cultures were shaken for 4 h; bacteria were then pelleted and resuspended in NENT buffer (20mM Tris (pH 8), 100mM NaCl, 1mM ethylenediaminetetraacetic acid (EDTA), 0.5% NP-40). Cell suspensions were sonicated and pelleted so that the supernatant could be collected.

[Page 60]

Briefly, 1x106 293FT cells (Invitrogen Corp, Carlsbad, CA, USA) were transfected with 2.25 μg of PAX2 packaging plasmid, 0.75 μg of PMD2G envelop plasmid and 3μg of pLKO.1 hairpin vector utilizing 30 μl of Fugene HD (Roche Applied Science, Indianapolis, IN, USA) in 10 cm plate. The lentivirus were collected and filtered (45um) after 48 hours. 2.5x105 T98G cells were plated in a multi 6 wells plate. The following day, the cells were transduced with 1 MOI of lentiviral particle in 10% FBS DMEM medium. After 3 days post-infection, the

[Page 61]

cells were selected with 2 μg/ml of puromycin (Sigma-Aldrich, St Louis, MO, USA) for 1 week.

[Page 65]

GST and GST-PIN1 proteins were produced in BL21 bacteria cells. Cells were grown to mid log phase and then induced to express protein by adding 0.25mM of isopropyl-1-thio-b-D-galactopyranoside (IPTG, Roche Applied Science, Indianapolis, IN, USA). The cultures were shaken for 4 h; bacteria were then pelleted and resuspended in NENT buffer (20mM Tris (pH 8), 100mM NaCl, 1mM ethylenediaminetetraacetic acid (EDTA), 0.5% NP-40). Cell suspensions were sonicated and pelleted so that the supernatant could be collected.

Anmerkungen

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

Sichter
(Graf Isolan), SleepyHollow02


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Letzte Bearbeitung dieser Seite: durch Benutzer:Singulus, Zeitstempel: 20141205190514

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