Fig. 3

EPE expression, facilitated by neurotrophin pro-domain processing, inhibits ERK nuclear translocation in COS7 cells. COS7 cells were transduced with either proBDNF-EPE, proNGF-EPE or GFP control LVs (MOI 5) and grown in DMEM medium supplemented with 10% FBS. 24 h prior to the experiment cells were serum starved (16 h, DMEM 1%FBS) and then either stimulated with TPA (200nM, 15 min), or left untreated (NT) as control. Nuclear and cytosolic ERK1/2 protein levels were evaluated by western blot. A: ERK1 nuclear translocation is presented as the ratio between ERK1 nuclear levels and ERK1 cytosolic levels, results were normalized to GFP NT sample (n ≥ 3, NT: con versus proBDNF-EPE, p = 0.0177, con versus proNGF-EPE, p = 0.0077. TPA: con versus proBDNF-EPE, p = 0.0014, con versus proNGF-EPE, p = 0.0006). B: ERK2 nuclear translocation is presented as the ratio between ERK2 nuclear levels and ERK2 cytosolic levels, results were normalized to GFP NT sample (n ≥ 3, NT: con versus proBDNF-EPE, p < 0.0001, con versus proNGF-EPE, p < 0.0001. TPA: con versus proBDNF-EPE, p < 0.0001, con versus proNGF-EPE, p < 0.0001). C: Immunoblots representing the levels of ERK1/2 in the cytosol and in the nucleus D: Phospho-Elk1 levels are presented as the ratio between phospho-Elk1 and total Elk1. Results were normalized to GFP NT sample (n ≥ 4, GFP NT Versus GFP TPA 200nM: p < 0.0001, TPA: GFP Versus proBDNF-EPE, p = 0.0231). E: Representative immunoblots of total and phosphorylated Elk1. F: Phosphorylated ERK1 levels are presented as the ratio between phospho-ERK1 and total ERK1. Results were normalized to GFP NT sample (n ≥ 3, GFP NT Versus GFP TPA 200nM, p < 0.0001, TPA: GFP Versus proBDNF-EPE, p = 0.0208). G: Phosphorylated ERK2 levels are presented as the ratio between phospho-ERK2 and total ERK2. Results were normalized to GFP NT sample (n ≥ 3, GFP NT Versus GFP TPA 200nM, p < 0.0001, TPA: GFP Versus proBDNF-EPE, p < 0.0001, GFP Versus proNGF-EPE, p < 0.0001). H: Representative immunoblots of pERK1/2 and tERK1/2. All data are presented as mean ± SEM