Fig. 2. Long-term activation of p42/p44 MAPKs induces their nuclear accumulation in the dephosphorylated form. Quiescent CCL39 fibroblast cells (A) or quiescent Raf-1:ER cells (B), were stimulated for the indicated times with 10% serum or 1 µM estradiol, respectively. 0 minutes corresponds to non-stimulated cells. Indirect immunofluorescence detection was performed with the monoclonal antibody anti-activated p42/p44 MAPKs from Sigma. (C) Quiescent CCL39 fibroblast cells were left non-stimulated (NS) (a,c) or stimulated with 10% serum (FCS) for 3 hours (b,d,e,f). Indirect immunofluorescence detection was performed either with the polyclonal anti-p42/p44 MAPKs antibody from UBI (green; a,b,e) or with the monoclonal anti-activated p42/p44 MAPKs antibody (red; c,d,f). For e and f, confocal microscopy was performed as described in Materials and Methods.