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Fig. 3. The coatomer subunit Ret2p binds to the GST-fusion peptides related to the cytoplasmic tail of GMT. The GST-fusion proteins were produced in E. coli and affinity purified by glutathione-Sepharose beads. The yeast lysate was prepared from the cells producing the 3HA-tagged Ret2p with glass beads and 0.5% Triton X-100 and used as the labelled coatomer. The beads were mixed with the lysate, incubated for 16 hours at 4°C and washed extensively, and the bound proteins were analysed by SDS-PAGE. The bound Ret2p was detected by immunoblot analysis with 12CA5 (top). The amount of GST-fusion peptide was shown by Coomassie Brilliant Blue staining (bottom). In lanes 1 and 14, the purified GST was used as the negative control. In lanes 2 and 15, the GST-fusion protein containing the Wbp1p peptide was used as the positive control. In lane 3, the GST-fusion protein containing the wild-type GMT C-terminal peptide was used.
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