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Fig. 9. Mon2p associates with the peripheral membrane protein Dop1p. (A) Dop1p-HA-mRFP is peripherally associated with membranes. JEY069 (MON2-GFP DOP1-HA-mRFP) cells (20 OD₆₀₀ units) were osmotically lysed and centrifuged at 13,000 g. The supernatant (S13) fraction was differentially treated as indicated, and further separated by ultracentrifugation at 100,000 g. The resulting high-speed pellet (P) and supernatant (S) fractions were analyzed for relative Dop1p-HA-mRFP content by western blotting. (B) Mon2p colocalizes with Dop1p. JEY069 cells were grown to mid-logarithmic phase at 26°C, shifted to 4°C for 10 minutes to minimize rapid movement of Golgi and endosomal elements, and observed by fluorescence microscopy. (C) Mon2p-GFP coimmunoprecipitates with Dop1p-HA-mRFP. Detergent-solubilized native lysates (50 OD₆₀₀ units) from JEY003 (MON2-GFP) and JEY069 were cleared at 16,000 g and subjected to immunoprecipitation using anti-HA antibody. Purified proteins were subjected to SDS-PAGE and western blotting analysis with anti-GFP and anti-HA antibodies. * indicates a possible breakdown product of Dop1p-HA-mRFP.

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