Figure 4

Differential signal processing results in differential activation of transcription factors. A20 cells were transfected with phosphatase specific siRNAs or mock siRNA, stimulated for 0, 30 and 60 minutes and were then stained with antibodies specific to transcription factors pp65, NFAT and p-c-jun followed by secondary antibody conjugated to Alaxa488. Cells were also stained with DAPI to locate the nucleus and were then monitored under Nikon TE 2000E microscope equipped with 60×/1.4 NA planapochromat DIC objective lens. Panel A, B and C shows representative fields for pp65, NFAT and p-c-jun respectively. Within each panel, column one shows antibody specific fluorescence, column two shows nuclear staining of the cells by DAPI, third column shows merging of the first two images (to see co-localization) and fourth columns shows white light image of the cells for which fluorescence were measured. The rows in each panel show various time points after stimulation of the cells. Panel D shows similar images for p-c-Jun under SHP1 knockdown condition. Images in column three, panel D bordered red (0 minutes and 60 minutes post stimulation) are enlarged below in Panel E for better depiction of visible differences in co-localization (see text for detail) in stimulated cells. Co-localization coefficients were calculated (for details see Additional Methods) for all the three transcription factors in the nucleus at every time points under all the perturbation conditions. Values measured from a minimum of 15 cells were taken to obtain average co-localization and they are plotted for all the transcription factors under various conditions (Panel F).