Figure 2

E2F4 is phosphorylated by ERK kinases. A. Subconfluent HIEC were serum-deprived for 36 h, treated or not (DMSO) during 10 min with 20 μM U0126 and then stimulated during 30 min with 5% FBS. Thereafter, cells were lysed and proteins were analyzed by SDS-PAGE (on 7.5% or 12% acrylamide gels) for Western blot analysis of the expression of phosphorylated ERK1/2, total ERK1/2, E2F4 and β-actin. B. E2F4 was immunoprecipitated from subconfluent serum-deprived and 30 min serum-stimulated HIEC. Beads containing E2F4 immune complexes were incubated with PP1 phosphatase for 30 min prior to Western blot analysis for E2F4 expression. C. E2F4 was immunoprecipitated from HIEC stimulated during 30 min with FBS in presence or absence of 20 μM U0126. E2F4 immune complexes were analyzed by SDS-PAGE for Western blot analysis with antibodies recognizing phosphorylated serine or E2F4. D. 293T cells were transfected with pCDNA3.1 containing or not HA-E2F4. 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 active recombinant ERK1 for 5 min. Radiolabeled proteins were separated on SDS-PAGE and autoradiographed or analyzed by Western blot for the expression of E2F4.