Undifferentiated and differentiated ESCs were lysed in a buffer containing 1 mM EDTA, 50 mM Tris-HCl (pH 7.5), 70 mM NaCl, 1 % Triton, and protease inhibitor cocktail (Sigma), and analyzed by western blot. The following primary antibodies were used: mouse Oct3/4 (1:1000 Santa Cruz Biotechnology, #sc-5279), rabbit Nanog (1:1000 Calbiochem-EMD Biosciences, #SC1000), mouse GAPDH (1:1000 Santa Cruz Biotechnology, #sc-32233), goat Sox1 (1:100 Santa Cruz Biotechnology, #sc-17318), rabbit Otx2 (1:500 Abcam, #ab114138), rabbit HMGA2 (1:500 Cell Signaling, #5269), and rabbit HMGA2 (1:500). GAPDH was used as an internal control. Antibody protein complexes were detected by HRP-conjugated antibodies (Anti-rabbit IgG, 1:10000, Amersham Pharmacia, #NA934V; Anti-mouse IgG, 1:5000, Amersham Pharmacia, #NA931V; Anti-goat IgG, 1:20000, Sigma-Aldrich, #A5420) and ECL (Amersham Pharmacia).
Navarra A., Musto A., Gargiulo A., Petrosino G., Pierantoni G.M., Fusco A., Russo T, & Parisi S. (2016). Hmga2 is necessary for Otx2-dependent exit of embryonic stem cells from the pluripotent ground state. BMC Biology, 14, 24.
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