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Fig. 2. The effect of targeted parvalbumin fusion proteins on testosterone-induced cytosolic and nuclear Ca²⁺ responses. Western blots of PV-DSR fusion proteins. Cells were transfected with expression vectors for PV-NLS (A), PV-NLS-CDEF (B) or PV-NES (C). Total cell lysates were separated by SDS-PAGE and parvalbumin protein was visualized with a monoclonal anti-PV antibody; ß-actin is shown as a loading control. In all three conditions the expression of PV was increased in the transfected cells, as compared with the untreated cells. Experiments are representative of three independent experiments. The subcellular distribution of the PV constructs was examined using confocal microscopy. Red indicates the location of DSR, blue indicates nuclear staining with TO-PRO3. (D) PV-NLS-DSR and (E) PV-NLS-CDEF-DSR (targeted to nucleus) are restricted to the nucleus. (F) Expression of PV-NES-DSR (targeted to cytosol) is uniformly distributed throughout the cytosol but excluded from the nucleus. (G) Cells transfected with the PV-NLS-DSR did not exhibit any nuclear Ca²⁺ increase after testosterone exposure, but the cells showed normal cytosolic Ca²⁺ oscillations. (H) In cells expressing PV-NLS-CDEF-DSR, a mutated PV that does not bind Ca²⁺, testosterone induced cytosolic and nuclear Ca²⁺ oscillations. (I) Cells expressing PV-NES-DSR did not exhibit any cytosolic or nuclear Ca²⁺ increase.

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