Figure 6.
Heat stress inhibits the degradation of EGFP bearing a ubiquitin-dependent degron, but not that of EGFP with a ubiquitin-independent degron. CHO cells were transiently transfected with plasmids encoding either GFPu or GFP-ODC. (A) Cells were lysed without any treatment (0h C), or incubated in the presence of cycloheximide for: 4 h at 37°C with MG132 (MG132), 30 min at 42°C followed by 3.5 h at 37°C (42°C), or 30 min at 37°C followed by 3.5 h at 37°C (mock). Total cell lysates were analyzed by Western blotting with anti-GFP antibodies. The amount of GFPu or GFP-ODC remaining after a 4-h chase in the presence of CHX under the indicated conditions was graphed as the fold increase relative to that obtained from CHX-treated mock-shocked cells. For each experimental condition, n = 3. (B) CHO cells transiently transfected with plasmids encoding either GFPu or GFP-ODC were “preshocked” by a 30-min incubation at 42°C or mock-shocked at 37°C and returned to 37°C for 3.5 h. The cells were then lysed without any treatment (0h C), or incubated for 4 h with CHX at 37°C and analyzed by Western blotting with anti-GFP antibodies. The amount of GFPu or GFP-ODC remaining after the CHX chase was calculated relative to the corresponding 0h C sample and was graphed relative to the value obtained from CHX-treated mock-shocked cells. For each experimental condition, n = 3.