ERK-specific gene silencing unmasks differential roles for ERK1 and ERK2 in cell signaling and proliferation. (a) Schematic representation (top) of the proviral vector form used in shRNA-mediated RNA interference. ΔU3, R and U5 constitute a chimeric long terminal repeat (LTR) of the HIV-1 5' LTR with a deletion in U3 abolishing LTR mediated transcription; SD and SA, splice donor and acceptor sites; ψ encapsidation signal including the 5' portion of the gag gene (GA); RRE, Rev-response element; cPPT, central polypurine tract; shRNA, small hairpin RNA; H1, human H1 promoter; mPGK, mouse phosphoglycerate kinase promoter; Puro, puromycin-resistance gene; WPRE, woodchuck hepatitis virus post-transcription regulatory element. The western blot (bottom) shows expression levels of ERK proteins in wild-type MEFs transduced with equal amounts of lentiviral vectors carrying the indicated knock-down (KD) shRNA cassette or the corresponding control sequence (ctr). α-tubulin was used as a loading control. (b) Wild type (+), ERK1 KD or ERK2 KD MEFs were serum starved for 24 h and then stimulated with 20% serum for 5, 10, 30, 60 and 120 min. Western blots were analyzed with anti-phospho-ERK and anti-ERK antibodies, as in Figure 1. (c) Bands from (b) were quantified and fold increases in phospho-ERK2 or phospho-ERK1 levels over total ERK2 or total ERK1 levels calculated. Mean ± SEM of three experiments is indicated. (d) Growth curve of wild-type, ERK1 and ERK2 KD fibroblasts and their corresponding controls, seeded in triplicate in the presence of 10% serum and 2 μg/ml puromycin and counted after the indicated times. The data are the mean of three independent experiments ± SEM.