Fig. 4

Only ERK1 protein and erk1 mRNA are expressed significantly in lizards. a siRNA sequences targeting anole lizard (A. carolinensis) erk1 mRNA (ERK1-A, ERK1-B and ERK1-C) and anole lizard erk2 mRNA (ERK2-A, ERK2-B and ERK2-C). In red, the nucleotides that differ when both erk1 and erk2 isoforms are aligned. b, c siRNA pools targeting erk1 or erk2 or control-siRNA were transfected three days prior to mRNA harvesting. b Absolute quantification of erk1 mRNA (left panel) or erk2 mRNA (right panel) after siRNA transfection, in A. sagrei and A. carolinensis lizard embryo fibroblasts (LEFs). erk1 or erk2 mRNA quantities were determined relatively to quantities of linearized plasmids harboring either A. carolinensis erk1 or erk2 cDNAs as described in materials and methods. To ease comparison between erk1 and erk2, data are expressed as percentage of siControl quantities. Bars represent mean +/− s.d. n = 4. c Western-blot analysis of samples from two independent transfections loaded on the same gel (Experiment 1 and 2) (insert) Fluorescence quantification of western blot. d Number of erk1 and erk2 mRNA molecules per 25 ng of total RNA from reptile samples: lizard (A. carolinensis), Crocodile (C. niloticus), turtle (T. scripta elegans) and mouse NIH3T3 cells. Bars represent means +/− s.d. n = 3 (e) Firefly luciferase activity driven by mouse and anole lizard ERK promoters (1 kb upstream from start ATG). Normalization by co-transfection of a plasmid expressing Renilla luciferase driven by the thymidine kinase promoter. Transfection was performed in A. carolinensis fibroblasts (left panel) or mouse NIH3T3 fibroblasts (right panel). Bars represent means +/− s.d. n = 4