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Fig. 5. Gyp5p-Myc and Gyl1p-HA interact with Sec4p-GFP. Lane 1, bud emergence; lane 2, small-budded cell; lane 3, cytokinesis. (A) Subcellular fractions obtained from 108 cells expressing Gyp5p-Myc, Gyl1p-HA and Sec4p-GFP were submitted to immunoprecipitation with anti-GFP antibodies, as described in Materials and Methods. Proteins bound to the agarose beads (Pellet), or remaining in the supernatant (Supernatant), were separated on SDS-PAGE and revealed with either anti-Myc, anti-HA or anti-GFP antibodies. One fifth of the total amount of proteins was loaded in supernatant lanes. In the control lanes, anti-GFP antibodies were omitted. (B) Yeast cells expressing Gyp5p-Myc, Gyl1p-HA and Sec4p-GFP were stained by immunofluorescence and examined with 3D deconvolution microscopy. Gyl1p-HA is stained in red, Sec4p-GFP is stained in green and Gyp5p-Myc is stained in blue. Images in the first row are projections of deconvoluted Z-series combining red, green and blue signals. White squares indicate the region magnified in the second row. Images in the second row are single sections shown with higher magnification. Bars, 1 µm.
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