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| Figure 2: The extraction protocol does not cause non-specific leakage of proteins into the extracellular fraction. (A), Wild-type cells expressing Scw11p- GFP were grown in YPKG for 3 days and examined for expression of proteins following glucose addition for 0 and 2 hours using Western blotting with anti-GFP antibodies. (B), Cells expressing Scw11p-GFP were glucose starved and taken for fluorescence imaging before extraction. Extracellular proteins were extracted and cells were visualized by fluorescence microscopy after extraction. (C), Cells expressing Scw11p-GFP were glucose starved and subjected to the extraction procedure. Following extraction, the distribution of Scw11p-GFP in the Intracellular (I) fraction and the Extracellular fraction (E) was examined by Western blotting. Wild-type cells that expressed Tps2p-GFP were examined for protein expression (D), GFP distribution before and after extraction by fluorescence microscopy (E), and the distribution in I and E fractions following extraction by Western blotting. (F), Wild-type cells expressing Sur7p-GFP were examined for expression (G), GFP distribution before and after extraction (H), and the distribution in the I and E fractions after extraction (I). |
