We have found that cGMP-dependent protein kinase (G-kinase) regulates transcription of the cfos gene apparently throught three different response elecments, i.e., a CRB, TRE and SRB. one previous publication indicates that G-kinase translocates to the nucleus in-human monocytes but this was not rigorously established. G-kinase does have a nuclear translocation sequence and we have evidence in G-kinase transfected cells by immunofluorescent microscopy that G-kinase does translocate to the nucleus when the cells are treated with a membrane-permeable cGMP analog. We aould now like to use confocal microscopy to obtain evidence that G-kinase is actually in the nucleus rather than around the nuclear envelope. We are staining the cells with a highly specific polyclonal Gkinase antibody and using an FITC-labeled secondary. In addition, we have now tagged our protein with the green fluorescent protein and there's a suggestion that the protein becomes intranuclear but unfortunately the staining is weak. We'd like to look at the samples with the confocaI instrument where we can amplify the signal more and ultimately, employ IvEM for high resolution localizations.
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