mESC Culture and Antibody Validation

We used the mESC line E14Tg2a in this study. Cells were cultured on 0.1% gelatin-coated (Millipore) plates in DMEM (Life Technologies) supplemented with 15% fetal bovine serum (Sigma), penicillin/streptomycin, nonessential amino acid, sodium pyruvate, GlutaMax, β-mercaptoethanol (Life Technologies), and 1000 U/mL LIF (ESGRO, Millipore) unless specified. Antibodies used in this study were 5hmC (Active Motif, 39769), Oct4 (Santa Cruz Biotechnology, sc-8628), Nanog (Bethyl Laboratories, IHC-00205), Sox2 (Millipore, Ab5603), Tet1 (Ito et al. 2010 (link)), Tet2 (described below), Tet3 (Gu et al. 2011 (link)), Zscan4 (Millipore, Ab4340), Suz12 (Cao et al. 2002 (link)), Ring1b (Cell Signaling, 5694), Ezh2 (Cell Signaling, 5246), and Kap1 (Cell Signaling, 4123). Tet2 antibody was generated using HIS-tagged recombinant Tet2 2-374 expressed in Escherichia coli and purified using Talon superflow metal affinity resin (Clontech). Rabbit immunization was carried out by Pocono Rabbit Farm and Laboratory, Inc. The antiserum was affinity-purified using immunization antigen. The specificity of the affinity-purified antibody was confirmed using extracts from the control and Tet2 knockdown mESCs.

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Lu F., Liu Y., Jiang L., Yamaguchi S, & Zhang Y. (2014). Role of Tet proteins in enhancer activity and telomere elongation. Genes & Development, 28(19), 2103-2119.

Publication 2014

gelatin-coated DMEM fetal bovine serum penicillin/streptomycin GlutaMax β-mercaptoethanol Nanog Zscan4 Suz12 Ring1b Talon superflow metal affinity resin

Amino acid Antibodies Antibody Antibody specificity Antigen Antiserum Cells Escherichia coli Ezh2 Fetal bovine serum Gelatin Immunization Kap1 Mercaptoethanol Mescs Metal Oct4 Penicillin Pyruvate Rabbit Resin Ring1b Sodium Sox2 Streptomycin Talon

Corresponding Organization :

Other organizations : Boston Children's Hospital, Howard Hughes Medical Institute, Harvard Stem Cell Institute, Harvard University

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Variable analysis

independent variables

0.1% gelatin-coated plates
DMEM supplemented with 15% fetal bovine serum, penicillin/streptomycin, nonessential amino acid, sodium pyruvate, GlutaMax, β-mercaptoethanol, and 1000 U/mL LIF

dependent variables

Expression of 5hmC, Oct4, Nanog, Sox2, Tet1, Tet2, Tet3, Zscan4, Suz12, Ring1b, Ezh2, and Kap1

control variables

Cells from the mESC line E14Tg2a

controls

Tet2 knockdown mESCs as a negative control to confirm the specificity of the Tet2 antibody

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