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Figure 4


Fig. 4. Vacuole targeting of Vac8 through the Src kinase SH4 domain. (A) Comparison of the SH4 sequence of Vac8 and the Src kinase. Alignment of the two SH4 domains is shown. (B) Localization of Src(1-16)-GFP by confocal fluorescence microscopy. The GFP-fusion protein was expressed from a plasmid in the vac8{Delta} background. Vacuoles were visualized with the lipophilic dye FM4-64. (C) Vacuole targeting of Vac8 through the Src kinase SH4 domain. Src-Vac8-GFP protein was expressed from a plasmid in vac8{Delta} cells and monitored by confocal microscopy in FM4-64 stained cells. Bars, 5 µm. (D) Subcellular localization of Src-Vac8. Fractionation was done as in Fig. 1C. Pellets and TCA-precipitated supernatant were analyzed by SDS-PAGE and western blotting with anti-Vac8, anti-Rpl13 and anti-Vti1 antibodies. Rpl13 is a ribosomal subunit (cytosolic marker), Vti1 is found on vacuoles (membrane marker). See Materials and Methods for details.





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