Fig. 8. Effect of cycloheximide on temperature-shifted GFP8 cells. (A) Cells, cultivated at 15°C, were either left untreated (lane 1), heat shocked and incubated at the conditions indicated (lane 2-4), or shifted to 32°C without heat shock (lane 5). After 6 hours, cells were extracted with detergent and the insoluble fractions analyzed by western blotting with anti-SFA. The strong decrease in the amount of SFA-GFP observed after a shift from 15 to 32°C (compare lanes 1 and 5) was partially inhibited by cycloheximide (lane 4). Furthermore, cells treated with cycloheximide (lane 4) contained less wild-type SFA in comparison with controls (lane 3 or 5). (B) Cells, cultivated at 15°C (lane 1), were shifted to 32°C for 6 hours either with (+, lane 3) or without (-, lane 2) cycloheximide. After removal of the cycloheximide (+ in brackets, lane 4), we observed a strong increase in the amount of SFA-GFP, and especially SFA. Temperature and time points are indicated (temp./hours). Anti-SFA was used for detection of SFA and SFA-GFP and similar amounts of cytoskeletons were loaded in lanes 3 and 4. (C) Fluorescence images of GFP8 cells corresponding to the experiment documented in the western blot shown in B. (1) GFP8 cells from a 15°C culture after 6 hours at 32°C; (2) as 1 but two hours later and centrifuged as the sample with cyloheximide; (3) GFP8 cells from a 15°C culture after 6 hours at 32°C in the presence of cycloheximide; (4) as 3, but two hours after cycloheximide has been removed. The cells had assembled long SFA-GFP fibers and some cells contained additional globular aggregates (insert). Bars, 10 µm (overviews) or 5 µm (insert).