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Fig. 5. Subcellular localization of HpPex20p. (A) Glucose/choline grown WT cells were disrupted using glass beads in buffer lacking (–) or containing (+) a protease inhibitor cocktail and incubated for 10 minutes at room temperature. Crude extracts prepared from cells that were immediately TCA precipitated upon harvesting were used as a control (TCA). HpPex20p levels were analysed by western blotting using anti-Pex20p antibodies. Equal amounts of protein were loaded per lane. These experiments revealed that HpPex20p is partially protected by the addition of the protease inhibitor cocktail (+). However, still approximately 90% is degraded relative to cells that were immediately TCA precipitated. (B) From glucose/choline-grown WT cells a post nuclear supernatant (PNS) was prepared in the presence of the protease inhibitor cocktail and subsequently centrifuged at 30,000 g. Western blot analysis revealed that HpPex20p is present in the organellar pellet. P3-30.000g pellet, S3-30,000 g supernatant. (C) Fluorescence microscopy of WT cells expressing HpPex20.eGFP and DsRed-SKL. This revealed that the green fluorescence was present in a spot at the same localization as the DsRed-SKL fluorescence.
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