FIGURE 2.
ERK mediates the down-regulation of OCT4A and induces its nuclear exclusion. A and B, NTera2 cells were treated with increasing doses of PD98059 (PD; A) or with 100 ng/ml FGF2 for the indicated times (B), and the relative protein levels of OCT4A and phospho-ERK were quantified. Lysates were analyzed by immunoblotting with the indicated antibodies. C, real-time PCR results confirmed the up-regulation of the key pluripotency-controlling genes Nanog, SOX2, and REX1 and the down-regulation of the differentiation-related genes BMP4, GATA6, and DLX5 after PD98059 treatment. D, real-time PCR results confirmed the down-regulation of the key pluripotency-controlling genes Nanog, SOX2, and REX1 and the up-regulation of the differentiation-related genes BMP4, GATA6, and DLX5 after FGF2 treatment. E, lysates of NTera2 cells transfected with control or MEK1CA plasmids were analyzed by immunoblotting using the indicated antibodies, and the relative protein levels of OCT4A and phospho-ERK were quantified. F, lysates of NTera2 cells were subjected to immunoblotting with the indicated antibodies after being transfected with a control vector or ERK1 and ERK2 siRNAs. G, NTera2 cells were serum-starved and then treated with FGF2 (100 ng/ml) for 30 min in the presence (+) or absence (−) of PD98059. Nuclear (N) and cytoplasmic (C) fractions were analyzed by immunoblotting with the indicated antibodies. The relative protein levels of OCT4A (normalized to α-tubulin (α-Tub) or poly(ADP-ribose) polymerase (PARP)) and phospho-ERK (normalized to ERK) were calculated. Graphs show the mean value of the representative results from three experiments (n = 3) with S.D. conducted in duplicate for each. An equal amount of lysates was loaded in each lane.