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Fig. 3. Exogenous GFP-ERK5 is localized in the cytoplasm. (A) HeLa cells were grown on coverslips, transfected with GFP-ERK5 construct (2.5 µg) or GFP-ERK5 (2.5 µg) together with either HA-WT-MEK5, HA-AA-MEK5 or HA-MEK5D (2.5 µg each) using the PEI method (Materials and Methods) and left untreated for additional 24 hours. This was followed by serum-starvation (0.1%) for 18 hours, after which the cells were left either unstimulated (NS) or stimulated with EGF (50 ng/ml, 30 minutes). Then, the cells were washed with PBS, fixed with PFA, and the GFP was visualized using a fluorescence microscope. (B) HeLa cells were grown in 6 cm tissue culture plates and transfected with GFP-ERK5 alone (4 µg) and together with either WT-MEK5 (WT, 4 µg) or HA-MEK5D (D, 4 µg). As above, the cells were left to recover, serum-starved, and either stimulated with EGF (50 ng/ml, 30 minutes) or left unstimulated (NS). Then the cells were harvested and subjected to western blotting with anti P-ERK5 and GFP antibodies.
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