Figure 3

Phosphorylation of E2F4 on serines 244 and 384 promotes its transcriptional activity. A. and B. 293T cells were transfected with pCDNA3.1 empty vector (EV) or encoded for HA-tagged- human wild-type (WT) E2F4 or E2F4 mutants as indicated. After 48 h, cell lysates were analyzed for the expression of E2F4 proteins. C. 293T cells were transfected with EV or encoding for HA-E2F4 WT, HA-E2F4 S244A, HA-E2F4 S384A or HA-E2F4 S244A/S384A. After 48 h, cells were lysed and immunoprecipitated with anti-HA antibody. Kinase assays were performed by incubating beads containing HA-E2F4 immune complexes with recombinant ERK1 for 5 min. Radiolabeled proteins were separated on SDS-PAGE and autoradiographed or analyzed for the expression of E2F4. D. 293T cells were co-transfected with DP2, thymidine kinase luciferase reporter with either empty vector, HA-wtE2F4, HA-E2F4 S244E, HA-E2F4 S384E or HA-E2F4 S244/S384E. pRL-SV40 Renilla luciferase reporter control vector was also co-transfected. Forty-eight hours following transfection, luciferase activity was quantified and normalized using the Renilla reporter, with the empty vector condition set at 1. A representative experiment of three experiments is shown. *Significantly different from control at p < 0.001, **Significantly different from WT at p < 0.002, ***Significantly different from WT at p < 0.001 (Student’s t test). E. HIEC grown on coverslips were transfected with either empty vector, HA-wtE2F4, HA-E2F4 S244E, HA-E2F4 S384E or HA-E2F4 S244/S384E. After 48 h, cells were analyzed by immunofluorescence for subcellular localization of HA-tagged E2F4 forms. Total cell number was determined using DAPI staining and cells exhibiting E2F4 forms into the nucleus or into the cytoplasm or in both compartments were counted. The percentage of cells exhibiting HA staining into the nucleus, into the cytoplasm or in both compartments was calculated and shown in the graph. Representative experiment is shown.