In vitro Methyltransferase Assay for β-catenin

In vitro methyltransferase assays were performed as described previously [19 (link), 20 (link)]. Briefly, recombinant GST-WT-β-catenin protein (#12-537; Millipore) was incubated with recombinant His-SMYD2 protein (in-house) and 2 μCi S-adenosyl-L- [methyl-³H]-methionine (Perkin Elmer, Waltham, MA) in a mixture of methylase activity buffer (50 mM Tris-HCl at pH 8.8, 10 mM DTT and 10 mM MgCl₂) for 2 h at 30°C. After denaturing, samples were separated by SDS-PAGE, blotted to PVDF membrane and visualized by MemCode Reversible Stain (Thermo Fisher Scientific, Waltham, MA) and fluorography.

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Deng X., Hamamoto R., Vougiouklakis T., Wang R., Yoshioka Y., Suzuki T., Dohmae N., Matsuo Y., Park J.H, & Nakamura Y. (2017). Critical roles of SMYD2-mediated β-catenin methylation for nuclear translocation and activation of Wnt signaling. Oncotarget, 8(34), 55837-55847.

Publication 2017

S-adenosyl-L- [methyl-3H]-methionine MemCode Reversible Stain

Assays Buffer Fluorography Membrane Methionine Methylase Mgcl2 Protein Pvdf Recombinant protein Sds page Stain Tris β catenin

Corresponding Organization : University of Chicago

Other organizations : RIKEN Center for Sustainable Resource Science, OncoTherapy Science (Japan)

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Variable analysis

independent variables

Recombinant GST-WT-β-catenin protein
Recombinant His-SMYD2 protein

dependent variables

Methylation of GST-WT-β-catenin protein

control variables

Methylase activity buffer (50 mM Tris-HCl at pH 8.8, 10 mM DTT and 10 mM MgCl2)
Incubation time (2 h)
Incubation temperature (30°C)
S-adenosyl-L-[methyl-3H]-methionine concentration (2 μCi)

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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