Protein kinase Cδ (PKCδ) translocates to the nucleus during tumor necrosis factor (TNF) treatment. (A) Confocal imaging of PKCδ localization. N27 cells were treated with TNF for 6 h and processed for immunocytochemistry using a rabbit polyclonal antibody that recognizes both the native and cleaved form of PKCδ. An Alexa-488 dye-tagged secondary antibody was used to visualize PKCδ (green), and the nucleus was stained using the TOPRO-3 dye (blue). In untreated controls (top panel), PKCδ was distinctly localized to the cytosol. In TNF-treated cells (lower panel), a distinct localization of PKCδ to the nucleus and the perinuclear region is seen. Scale bars are 20 microns. High magnification images (right panel) of PKCδ immunofluorescence staining in N27 cells showing PKCδ accumulation within the nucleus and at distinct spatial locations at the perinuclear region following TNF treatment (red arrows). Representative images are shown; experiments were repeated three times using different secondary antibodies for validation. (B) Fluorescence spatial intensity plots along a representative XY plane for PKCδ localization (green channel) and the nucleus (blue channel) were obtained using the Nikon EZC1 image analysis software. Control images show a distinct spatial localization of green and blue fluorescence intensities in the XY plane indicative of cytosolic PKCδ localization. In TNF-treated cells a spatial colocalization of green and blue fluorescence signals is evident indicating nuclear translocation of PKCδ.