Fig. 3. Mutation of E248 and D249 prevents ERK's NTS phosphorylation and nuclear translocation. (A) Mutation of D248 and E249 inhibits nuclear translocation of exogenous ERK2. CHO cells were co-transfected with the following GFP tagged plasmids: ERK2-WT (SPSQED), ERK2-EPEQED, ERK2-SPSQAA (SQAA), or ERK2-EPEQAA (EQAA) and RFP-MEK1. Thirty-six h after transfection the cells were serum starved and either stimulated with TPA (250nM, 15 min) or left untreated (NS). The cells were stained by DAPI and visualized by using fluorescent microscope. Scale bar - 15 µm. (B) Cells were treated as describe in A, and then fractionated to cytosolic/nuclear fractions and subjected to Western blot analysis. Tubulin and histoneH1 serve as purity and loading markers. P-ERK as well as ERK and MEK expression were also detected using whole cell extracts (lower blots). The bar-graph represents densitometric analysis of nuclear ERK (three experiments). * P<0.05. (C) Mutation of D248 and E249 inhibits ERK's NTS phosphorylation. 293T cells were transfected with the indicated GFP tagged plasmids, serum-starved and stimulated as indicated. Cell extracts were subjected for Western blot analysis with the indicated Abs.