Figure 6
Spry1 decreases wild type E1A 13S induced gene expression but not ΔNE1A 13S transactivation through ERK1/2 kinase phosphorylation. (A) HeLa cells were co-transfected with the reporter construct SRE-Luc (1 μg) and plasmids (0.5 μg) expressing Spry1 and/or E1A13S or ΔNE1A13S as indicated. Empty expression vectors were added to keep the amount of the transfected DNA constant. After transfection cells were left serum-deprived for 24 h and then incubated with 20 ng/ml bFGF in DMEM. Serum stimulation was performed for the indicated times (white, 1 h; cross line, 5 h; horizontal line, 7 h; dots, 9 h). The promotor activity of the reporter gene in the presence of empty vectors was set as 1. Data represents mean ± S.E.M. of n ≥ 4. * p < 0.05 (B) Expression of Spry1 and E1A13S in the lysates was confirmed in Western blot assays. (C) HeLa cells expressing Spry1 and/or E1A13S or ΔNE1A13S were serum starved overnight, followed by incubation for 1 h with 20 ng/ml bFGF in DMEM. Cells were lysed and lysates were subjected to SDS-PAGE and analyzed by immunoblotting. The membranes were incubated with antibodies directed against phosphorylated ERK1/2 (phospho-ERK1/2), unphosphorylated ERK1/2 (ERK1/2) and antibodies directed against Spry1 and E1A13S.