Fig. 3. Src activates the ERK5 pathway. (A) Activated Src (Y527F) was transfected together with different combinations of wild-type and mutant ERK5 and/or MEK5, the MEF2D luciferase reporter and a ß-galactosidase plasmid for normalisation. Results are the mean±s.e. of eight independent experiments. The decrease in luciferase activation in the Y527F Src + ERK5AEF sample was statistically significant (*) by the Student's t-test (P=0.01). Arrows indicate nuclei of microinjected cells. (B) Activated Src causes translocation of ERK5 from the cytoplasm to the nucleus. NIH3T3 cells were microinjected at 5:1 ratio with empty vector and an EGFP expression plasmid (upper panels) or activated Y527F Src and EGFP (lower panels). Cells were then stained with ERK5 antibody to detect endogenous ERK5. Src protein expression was inferred by GFP fluorescence. Arrows indicate nuclei of microinjected cells. (C) NIH3T3 cells were microinjected with HA-MEK5DD and stained with anti-HA and anti-ERK5 to detect endogenous ERK5. (D) ERK5 is present in the nucleus of Src-transformed cell. Different v-Src transformed NIH3T3 clones were serum starved for 24 hours and then stained as in B. Bar, 20 µm.