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Molecular Profiling of Cellular Pathways

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Whole cell lysis, subcellular fractionation, western blot and immunoprecipitation were performed as described [17 (link), 44 (link)]. Quantification of the band density of target proteins in western blot experiments was analyzed by free software ImageJ. The following antibodies were used in western blotting and immunoprecipitation: EZH2, phospho-GSK3β Ser9, trimethyl-H3K27, and histone H3 (Cell Signaling Technology); GSK3β (BD Biosciences); dephospho-β-catenin (Ezno Life Sciences); Tubulin and Actin (Sigma); Lamin B1 (Abcam); Myc and HA (Roche). The mouse phospho-EZH2 Thr367 antibody was produced against the synthetic peptides SRPS(pT)PTINVLESKD at China Medical University Hospital in Taiwan. The synthetic peptides were obtained from LifeTein LLC. Total RNA was extracted from cells using TRIzol. Quantitative real-time PCR (qRT-PCR) was performed using SYBR Green dye on a Bio-Rad PCR machine. The primer sequences used for analysis of HOXA gene expression was listed in Supplementary Information Table S1.

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Ko H.W., Lee H.H., Huo L., Xia W., Yang C.C., Hsu J.L., Li L.Y., Lai C.C., Chan L.C., Cheng C.C., Labaff A.M., Liao H.W., Lim S.O., Li C.W., Wei Y., Nie L., Yamaguchi H, & Hung M.C. (2016). GSK3β inactivation promotes the oncogenic functions of EZH2 and enhances methylation of H3K27 in human breast cancers. Oncotarget, 7(35), 57131-57144.

Publication 2016

phospho-GSK3β Ser9 GSK3β Tubulin and Actin Lamin B1 SYBR Green dye PCR machine

Actin Antibodies Antibody Cell Ezh2 Fractionation Gene expression analysis Gsk3β Histone h3 Immunoprecipitation Lamin b1 Mouse Peptides Primer Proteins target Quantitative real time pcr Srps Sybr green Trizol Tubulin Western blot β catenin

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Variable analysis

independent variables

Whole cell lysis
Subcellular fractionation
Western blot
Immunoprecipitation

dependent variables

Quantification of the band density of target proteins in western blot experiments

control variables

Antibodies used in western blotting and immunoprecipitation: EZH2, phospho-GSK3β Ser9, trimethyl-H3K27, histone H3, GSK3β, dephospho-β-catenin, Tubulin, Actin, Lamin B1, Myc, HA
Production of mouse phospho-EZH2 Thr367 antibody against synthetic peptides
RNA extraction using TRIzol
Quantitative real-time PCR (qRT-PCR) using SYBR Green dye on a Bio-Rad PCR machine
Primer sequences used for analysis of HOXA gene expression

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