FIG. 3.
Ectopic AKT1 activity blocks the activation and nuclear accumulation of GSK3β and c-myc T58 phosphorylation following LIF withdrawal. (A) Experimental scheme for panels B through F, involving a mESC line expressing a myristoylated form of AKT1 fused to the steroid binding domain of the ER (myr.AKT1-ER). The construct introduced expresses eGFP from an internal ribosome entry site (IRES) linked to the myr.AKT-ER cassette (28). (B) Cell lysates from mESCs (plus LIF) or cells cultured in the absence of LIF with (+) or without (−) 4OHT (1 μM) for 1 to 4 days (see panel A) were resolved by polyacrylamide gel electrophoresis and probed as indicated. (C through F) Levels of myr.AKT1-ER and its activity status were determined with antibodies for pan-AKT1 and AKT1pS473, respectively. mESCs (plus LIF) and cells cultured in the absence of LIF with or without 4OHT were probed with antibodies as indicated. Expression of myr.AKT1-ER was established by eGFP fluorescence from a linked IRES.