Gsk-3 does not mediate its effects via general translational machinery or the 5′UTR of Nanog.

<p>(<b>A</b>) E14tg2a wild-type (WT) and Gsk-3α/ß double knockout (DKO) ESCs were cultured in the presence of LIF and serum. Gsk-3 inhibitor 1 m was added to WT cells at 2 µM and protein samples taken at the times indicated. Cell lysates were blotted with the antibodies indicated. (<b>B</b>) WT and Gsk-3 DKO ESCs were grown in N2B27 plus BMP4 and LIF. WT were treated with 2 µM 1 m or 3 µM CHIR for 8 and 24 hours before lysis. Immunoblotting was performed with antibodies against pT389 S6K1 and Gapdh. (<b>C</b>) E14tg2a ESCs were grown overnight in the presence of MEK inhibitor (PD) before being treated with 3 µM CHIR or 2 µM 1 m for the times indicated. ESCs were also grown in either CHIR or 1 m alone overnight prior to preparation of cell lysates. Immunoblotting to detect phosphorylation of T389 on S6K1 and Gapdh were performed. (<b>D</b>)(<b>i</b>) The 215bp 5′UTR sequence of the mouse Nanog gene (RefSeq NM_028016). Polypyrimidine tracts, shown in blue, were identified at bp positions 122, 137 and 191, with the tract at 137 containing 1 CCT motif and the tract at position 191 containing 2 CCT motifs (in bold font). One potential uORF, underlined, with an ATG start codon was identified between bp133 to 156. WT E14 ESCs were transfected with (<b>ii</b>) pGL3’ or pGL3’-Nanog-5′UTR in the presence of pRenilla luciferase plasmid or (<b>iii)</b> pGL3-MCS, pGL3-Nanog-3′UTR R1 or pGL3-Nanog-3′UTR R2 in the presence of pRenilla luciferase plasmid. After 24 h, transfected cells were treated with vehicle alone (C) or 2 µM 1 m. Cells were harvested at 4, 8 and 16 h and Firefly luciferase activity determined and normalised relative to Renilla luciferase activity. Luciferase activity is expressed as fold-change over pGL3’ (<b>ii</b>) or pGL3-MCS (<b>iii</b>) controls (DMSO) for a given time point. Data are the average and S.E.M. of three independent experiments.