Figure 2: The EPE peptide prevents interaction of ERK1 with importin7 and activation of nuclear ERK1/2 targets.

(a) EPE peptide prevents ERK1/2–importin7 interaction. HeLa cells were grown in full medium and then serum starved (0.1%, 16 h), pretreated either with EPE peptide, scrambled peptide (10 μM, 2 h) or DMSO control (0.1%), and then stimulated with tetradecanoyl phorbol acetate (TPA; 250 nM, 15 min). ERK1 was precipitated from cellular extracts using αgERK1 Ab; importin7 co-IP with ERK1 was detected by western blotting with αimportin7 Ab. The closest molecular weight marker (kDa) is shown on the right of all relevant panels. (b) EPE peptide prevents Elk1, and to a lesser extent ERK1/2’s NTS, but not RSK1 phosphorylation. T47D, HeLa, MDA-MB-231 and A2352 cells were grown in full medium and then serum starved (0.1%, 16 h), pretreated either with EPE peptide, scrambled peptide (10 μM, 2 h) or DMSO control (0.1%), and then stimulated with TPA (250 nM, indicated times). Cell extracts were subjected to western blot analysis with the indicated Abs. (c) EPE peptide inhibits phosphorylation of c-Myc and Elk1 in B-Raf melanomas. A2158 and A2352 cells were grown in full medium and then serum starved (0.1%, 16 h), treated with wither scrambled or EPE peptide (10 μM, 2 h), and then stimulated with the indicated concentrations of TPA for 15 min. Cell extracts were subjected to western blot analysis with the indicated Abs. (d) Quantification of some of the results in b,c. The bars represent fold difference between scrambled and EPE peptides in the phosphorylation of the indicated proteins after TPA stimulation. The results represent the average and standard errors of two or three experiments. (e) EPE peptide does not affect short-term AKT phosphorylation. The same extracts described in b were subjected to western blot analysis using anti-pAKT and gAKT Abs. (f) The EPE peptide does not affect long-term AKT phosphorylation. HeLa cells were treated either with EPE, scrambled peptide (10 μM) or DMSO control (0.1%) for the indicated times after which they were subjected to western blot analysis using anti-pAKT and gAKT Abs. The experiments were reproduced 2–4 times.