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Fig. 5. Inhibition of both the ERK1/2 and ERK5 pathways is required to restore the cytoskeleton in Src-transformed cells. (A) Src-transformed NIH3T3 cells (D4F9-ac2M) were treated for 24 hours with PD184352 or solvent alone then fixed and stained with Texas Red-phalloidin to visualise filamentous actin and a vinculin antibody to visualise focal adhesions. (B) Lysates of Src-transformed NIH3T3 cells treated for up to 48 hours with PD184352 (1=1 µM and 10=10 µM PD184352) or solvent alone (D=DMSO) were western blotted with antibodies against activated dually phosphorylated ERK1/2 and ß-tubulin and pan-ERK as loading controls. (C) v-Src transformed NIH3T3 cells (D4F9-ac2M) were microinjected with expression vectors for HA-MEK5AA, Flag-ERK5AEF or MEK1A. After 24 hours, cells were fixed and stained for HA-MEK5AA and Flag-ERK5AEF with HA (12CA5) and Flag (M2) directed antibodies. MEK1A expression was detected with a MEK1 antibody. Polymerised actin was stained with Texas Red-phalloidin. Bar, 20 µm.
