Immunolocalization of phospho-ERK 1/2 within GT1 cells. Infected and uninfected GT1 cells, both D18 and quinacrine treated and untreated, were fixed and immunostained with an antibody specific for the phosphorylated (Thr202/Tyr204) form of ERK 1/2 (in green); nuclei were counterstained with DAPI (in blue). Merged images are shown on the left. In ScGT1 phospho-ERK appears prevalently localized within the cytoplasm, whilst in GT1 cells the phospho-ERK staining is equally distributed between the cytosolic and the nuclear compartments (panel A). The treatments with both Fab D18 and quinacrine induce the active form of ERK to localize preferentially within the nuclear compartment in ScGT1, but have opposite effects on the phospho-ERK distribution in uninfected GT1 (panel A). As positive control, cells were treated with TPA (12-O-Tetradecanoylphorbol-13-Acetate) diluted in DMSO at the concentration of 200 nM for 30 minutes. As negative control, U0126 (10 μM in DMSO) was used, treating cells for 1 hour (panel A). Images are representative of at least three independent experiments of immunostaining. Scale bars, 20 μm. The quantitative analysis was carried out measuring the average intensity of fluorescence in a region of interest (ROI) of 144 pixels, both in the nuclear and cytoplasmatic regions. The histograms (panel B) show the ratio between the average intensity of fluorescence of phospho-ERK signal in the cytosol, over the average intensity of the same signal in the nucleus. More than 100 cells were analyzed for each condition. Statistics were performed using Student's T-test on a set of three independent experiments; *** P < 0.001.