Fig. 3.

Influence of DUSP knock-down on the time course of EGF-stimulated ERK activation. HeLa cells were transfected with control siRNA (Ctrl., all panels) or with a combination of siRNAs targeting the nuclear-inducible DUSPs 1, 2, 4 and 5 (NI DUSP KD, upper panels), the cytoplasmic ERK-directed DUSPs 6, 7 and 9 (Cyt. ERK DUSP KD, middle panels) or the JNK/p38 DUSPs 10 and 16 (JNK/p38 DUSP KD, lower panels) and then kept in medium with 0.1% FCS for 16 h prior to stimulation (with 10 nM EGF for 0 or 2–14 min), fixation, staining and imaging as above. The left panels show time course in cells stimulated without MEK inhibitor, whereas the right panels show time courses with PD184352 (10 μM) added 6 min after the stimulus (horizontal bar). Whole cell ppERK1/2 measures were normalized to internal control values (6 min ppERK1/2 levels in cells with control siRNA) and the data shown are means ± SEMs (n = 3) pooled from 3 separate experiments each with duplicate or triplicate wells. *P < 0.05, **P < 0.01 for comparison of control and test siRNAs. Rates of ERK inactivation after MEK inhibition were estimated (assuming exponential decay after MEK addition). The half-time was 2.61 ± 0.30 min (n = 3) in cells with control siRNA and this was not significantly different (P > 0.05) to the half times in cells with siRNAs targeting any of the DUSP families.