Fig. 7. FM4-64 uptake, and Ste6p internalization and degradation, were inhibited in an OSH temperature-sensitive mutant. (A) Protein immunoblots of hemagglutinin (HA) epitope-tagged Ste6p expressed in osh osh4-1 cells (CBY966) and its congenic wild-type Osh⁺ parent (CBY968), and las17 cells (CBY1024) and its congenic wild-type LAS17 parent (CBY1026). In each of these strains, the stability of Ste6p was analyzed by adding cycloheximide to stop protein synthesis and then removing, at the times indicated, equal volumes of cell culture incubated at 37°C. Extracted proteins were separated on SDS gels and analyzed by immunoblot with antibodies that recognized the HA epitope. Note that in the wild-type strains, Ste6p-HA degradation occurred after 60 minutes but in the OSH temperature-sensitive mutant and las17 strain Ste6p-HA persisted beyond 90 minutes. All strains were incubated for 1 hour at 37°C before the addition of cycloheximide. (B) Wild-type cells (Osh⁺; SEY6210), osh osh4-1 (CBY926) and osh OSH4 (CBY924) cells were incubated for 30 minutes at 37°C, then treated with a short pulse of FM4-64 and chased with fresh medium. Cells were viewed by fluorescence microscopy 30 minutes after the FM4-64 pulse/chase (P/C) and photographs were taken with equal exposures.