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Fig. 3. 14-3-3 and CaM regulate the subcellular distribution of Kir/Gem and Kir/Gem-mediated changes in cell morphology. (A and B) COS-1 cells were transfected with cDNAs for wt or mutated Kir/Gem, either alone, or together with GST–14-3-3, GST–14-3-3 K49E or GST–14-3-3 dimerization mutant. Cells were processed for immunofluorescence microscopy using Myc and GST antibodies to label Kir/Gem (red) and GST–14-3-3 (green), respectively. Areas of colocalization are in yellow in the merged image. Nuclei were stained with Hoechst 2022 (A and B, panel b). (C) Quantification of the 14-3-3-mediated cytosolic relocalization of Kir/Gem. 150-200 transfected cells were randomly selected and analyzed in 3-5 independent experiments. The fraction of cells showing efficient (black bars), partial (gray bar) or no (white bars) nuclear clearance is plotted. (D) Quantification of the 14-3-3-mediated cytosolic relocalization of Kir/Gem. Transfected cells were subjected to subcellular fractionation to obtain nuclear (N) and cytoplasmic (C) fractions. Kir/Gem in the different fractions was detected by SDS-PAGE and western blot analysis (Fig. S1 in supplementary material). The bands were scanned and the fraction of total Kir/Gem recovered in the nuclear and cytoplasmic fraction of three independent experiments is plotted. (E) Quantification of the Kir/Gem-induced morphological changes. 150-200 transfected cells were randomly selected and analyzed in 3-5 independent experiments. The fraction of cells showing one or more dendrite-like extensions, defined as thin protrusions of at least half a cell diameter, is plotted.
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