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Fig. 4. Stable overexpression and activation of GFP-MSK1 in C3H 10T{gamma} cells. (A) Quiescent wild-type (panels i-vi) or GFP-MSK1-overexpressing (panels vii-xii) cells were stimulated with 25 ng/ml anisomycin (sAn) for 45 minutes (panels iv-vi, x-xii) or left untreated (panels i-iii, vii-ix). Cells were fixed, permeabilised and processed for immunofluorescence as described in Materials and Methods. Cells were stained with rabbit anti-GFP primary and anti-rabbit-IgG-Alexa-488 secondary antibodies. Nuclei were stained with DAPI. Cells were examined by conventional fluorescence microscopy for Alexa-488/GFP- (green) and DAPI- (blue) fluorescence. Merged images are presented in the right column of images. Scale bar, 20 µm. (B) Quiescent wild-type or GFP-MSK1-overexpressing cells were stimulated with 100 nM TPA for 30 minutes (lanes 2, 4) or left untreated (lanes 1, 3). Cells were lysed and a fraction containing soluble nuclear and cytoplasmic proteins was obtained (see Materials and Methods). Proteins were separated by 10% SDS-PAGE and western blotted with anti-GFP (panel i), anti-MSK1 (panel ii) and anti-ERK (panel iii) antibodies. (C) Quiescent wild-type (lanes 1-9) or GFP-MSK1-overexpressing (lanes 10-18) cells were stimulated with 50 ng/ml EGF (lanes 2, 3 and 11, 12), 25 ng/ml sAn (lanes 4-6, 13-15) or 100 nM TPA (lanes 7-9, 16-18) for the times indicated. Cells were lysed and high-salt-extracted (see Materials and Methods) proteins were separated either on standard 10% SDS-PAGE (panel ii) or in modified gels that enhance phosphorylation-dependent electrophoretic retardation (panel i) (see Materials and Methods). Western blots were carried out with anti-MSK1 (panel i) and anti-ERK (panel ii) antibodies. (D) Quiescent wild-type (columns 1-3) or GFP-MSK1-overexpressing (columns 4-6) fibroblasts were stimulated for 30 minutes with 25 ng/ml sAn (columns 3, 6), for 20 minutes with 100 nM TPA (columns 2, 5), or left untreated (columns 1, 4). Cell lysates were immunoprecipitated with anti-GFP antibody, and immunoprecipitates assayed for kinase activity towards Crosstide peptide substrate as described in Materials and Methods. Assays were performed in triplicate on independent extracts. Error bars represent the standard deviation of the mean.