Fig. 2. Sly1^ts protein localizes on the membrane as does the wild-type Sly1 protein. The C-terminally myc-tagged Sly1, Sly1-20 and Sly1^ts proteins were produced in the sly1^ts strain from CEN plasmids. Cells containing Sly1-6myc (lanes 1-3), Sly1-20-6myc (lanes 4-6) and Sly1^ts-6myc (lanes 7-9) were lysed by vortexing with glass beads and were subjected to centrifugation at 1,000 g for 5 minutes to remove unbroken cells and debris. The supernatant was fractionated by differential centrifugation at 10,000 g for 10 minutes and 100,000 g for 1 hour at 4°C. Aliquots of the fractions were subjected to SDS-PAGE and western blot analysis with antibodies against the localization marker proteins shown to the left.