Fig. 5. (A) Dynamics of MEK phosphorylation, determined by western blotting, in RTgill-W1 cells exposed to 30 nmol l^–1 EGF for up to 60 min. Figures below the blot indicate changes in pMEK abundance, expressed relative to controls at time zero, and denote means ± s.e.m. of four independent experiments. c60, controls incubated in standard conditions for 60 min. (B) Cellular distribution of pMEK in RTgill-W1 cells incubated in standard conditions or exposed to EGF for 5 min. The right image shows cells pre-incubated with leptomycin B (LB) for 60 min prior to addition of EGF. Cells were stained with an antibody against the dually phosphorylated form of MEK and a FITC-labelled secondary antibody and were then examined by confocal laser scanning microscopy. Scale bar, 20 µm (valid for all three images). (C) Quantitative estimation of nuclear/cytoplasmic distribution of phosphorylated MEK, expressed as nuclear/cytoplasmic ratio, in RTgill-W1 cells exposed to EGF with or without prior pre-incubation with LB. Values are means ± s.e.m. of at least 14 cells from three independent cultures. For both treatments, nuclear/cytoplasmic ratio is significantly decreased at all time points compared to the value at time zero (P<0.05).