Fig. 5. Control experiments reveal the specificity of PKC translocation. (Ai) Treatment of osteoclast-like cells with vehicle alone had no effect on the distribution of PKC-EGFP, with unchanged localization in the cytosol. Plot at right illustrates the time course of membrane localization, with no change in response to vehicle. Test agents were applied at the time indicated by the vertical broken lines. Data shown are representative of six independent experiments. (Aii) An osteoclast-like cell expressing EGFP alone exhibited fluorescent label in both the cytosol and nuclei, in contrast to the localization of EGFP-tagged PKC (Ai). BzATP caused no change in distribution of EGFP, as quantified on the right. Data shown are representative of three independent experiments. (Aiii) Treatment with PMA (10 µM) caused persistent translocation of PKC-EGFP from the cytosol to the membrane (data shown are representative of eight independent experiments). (B) Images show an x-y section and reconstructed z-stack of an osteoclast-like cell, revealing that PMA induced PKC translocation to the basolateral membrane, similar to that seen in response to BzATP. Images labeled `After PMA' were obtained at 120 seconds following the addition of PMA. This osteoclast also exhibited a prominent vacuole in the centre of the cell. Notably, PKC did not translocate to the vacuolar membrane, as both the x-y and x-z projections show.