Fig. 4

A–D: Unchanged nucleo-cytoplasmic translocation. A–C HeLa cells were transfected with expression plasmids coding for either GFP- or SNAP-tagged mutants of STAT3 and stimulated for indicated times with 50 ng/ml of recombinant IFNγ. Fluorescence micrographs show unaltered nuclear accumulation of D427H-GFP and D427H-SNAP as compared to the hyper-phosphorylated F174A mutant, which served as a positive control. Histograms show the quantification of nuclear STAT3-GFP/-SNAP as a ratio of total cellular GFP/SNAP intensity from three independent experiments (n = 3). D Indirect immunofluorescence using an antibody against the phospho-tyrosine 705 residue in STAT3 confirms the translocation of phospho-STAT3 in response to IFNγ stimulation in transfected HeLa cells. GFP- and SNAP-tagged STAT3 variants were transfected in HeLa cells which were subjected to stimulation with 50 ng/ml recombinant IFNγ for the indicated times. These cells were fixed and stained with an antibody against phospho-STAT3, followed by a secondary antibody (Cy3 (red) for cells expressing STAT3-GFP and Cy2 (green) for cells expressing STAT3-SNAP). Means ± standard deviations from n = 20 cells with *p ≤ 0.05