Molecular Characterization of ERβ Regulation

Mission shRNA against ERβ was obtained from Sigma–Aldrich. Expression plasmid for HA-tagged mERβ (pSSH25-mERβ) was constructed by inserting PCR-amplified mERβ cDNA into pSHH25 [56 (link)] using XhoI and BlgII restriction sites. pSSH25-TDG for mammalian expression of HA-fused TDG [56 (link)], pPRS220 for yeast expression of Gal4-AD-fused TDG [46 (link)], 3× ERE-luc [47 (link)], and pSG5hERβ [57 (link)] used in luciferase assays have been published. pRL-TK for normalisation of luciferase activity was purchased from Promega. GST-ERβ was constructed by cloning cDNA encoding for human ERβ into pGEX-6P-3 (GE Healthcare) using BamHI and XhoI restriction sites. pACT2-ERβ was obtained by cloning cDNA encoding for human ERβ into pACT2 (Clontech) using SmaI and XhoI restriction sites. Antibodies used were as follows: rat monoclonal anti-HA (3F10) from Roche Applied Science, rabbit monoclonal anti-ERβ (05-824), rabbit polyclonal anti-H3K4m2 (07-030), and rabbit polyclonal anti-H3K27m3 (07-449) from Millipore, rabbit polyclonal anti-H3K9m3 (060-050) from Diagenode, and mouse monoclonal anti-Hsp90 (F-8) from Santa Cruz Biotechnology, Inc.

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Liu Y., Duong W., Krawczyk C., Bretschneider N., Borbély G., Varshney M., Zinser C., Schär P, & Rüegg J. (2016). Oestrogen receptor β regulates epigenetic patterns at specific genomic loci through interaction with thymine DNA glycosylase. Epigenetics & Chromatin, 9, 7.

Publication 2016

pRL-TK pGEX-6P-3 pACT2 rat monoclonal anti-HA (3F10)

Antibodies Assays Cdna Hsp90 Human Luciferase Mammalian Mouse Plasmid Promega Rabbit Shrna Smai Yeast

Corresponding Organization :

Other organizations : Shanghai Medical College of Fudan University, University of Basel, Bayer (Germany), Karolinska Institutet

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Variable analysis

independent variables

ShRNA against ERβ
Expression plasmid for HA-tagged mERβ (pSSH25-mERβ)
Expression plasmid for HA-fused TDG (pSSH25-TDG)
Expression plasmid for Gal4-AD-fused TDG (pPRS220)
3× ERE-luc reporter
PSG5hERβ expression plasmid
GST-ERβ fusion protein
PACT2-ERβ expression plasmid

dependent variables

Not explicitly mentioned

control variables

PRL-TK plasmid for normalization of luciferase activity

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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