Protein extraction and western blot analysis

Whole-cell extracts were prepared with transgenic buffer (20 mM Tris-HCl at pH 7.5, 137 mM NaCl, 1mM EGTA, 1% Triton X-100, 10% glycerol, 1.5 mM MgCl₂) supplemented with protease inhibitor cocktail (Sigma, P2714). Nuclear extracts were prepared as described previously (van den Berg et al. 2010 (link)). Western blotting was carried out according to a standard protocol (see the Supplemental Material). The antibodies used were anti-Flag (Sigma, F1804), anti-Elf5 (Santa Cruz Biotechnology, sc-9645), anti-Eomes (Abcam, ab23345; R&D Systems, MAB 6166), anti-Tfap2c (R&D System, AF5059), anti-tubulin (Abcam ab6160), and ImmunoCruz IP/WB Optima System C (sc-45040), E (sc-45042), or A (sc-45038).

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Latos P.A., Sienerth A.R., Murray A., Senner C.E., Muto M., Ikawa M., Oxley D., Burge S., Cox B.J, & Hemberger M. (2015). Elf5-centered transcription factor hub controls trophoblast stem cell self-renewal and differentiation through stoichiometry-sensitive shifts in target gene networks. Genes & Development, 29(23), 2435-2448.

Publication 2015

protease inhibitor cocktail anti-Elf5 ab23345 MAB 6166

Antibodies Buffer Cell extracts Egta Glycerol Mgcl2 Nacl Protease inhibitor Transgenic Tris Triton x 100 Tubulin

Corresponding Organization :

Other organizations : Babraham Institute, University of Cambridge, Osaka University, University of Toronto

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Variable analysis

independent variables

Transgenic buffer composition (20 mM Tris-HCl at pH 7.5, 137 mM NaCl, 1mM EGTA, 1% Triton X-100, 10% glycerol, 1.5 mM MgCl2)

dependent variables

Protein expression levels of Flag, Elf5, Eomes, Tfap2c, and tubulin measured by Western blotting

control variables

Protease inhibitor cocktail (Sigma, P2714) added to the transgenic buffer
Nuclear extracts prepared as described previously (van den Berg et al. 2010)

controls

Positive control: ImmunoCruz IP/WB Optima System C (sc-45040), E (sc-45042), or A (sc-45038)

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