Fig. 3.

NEDD8 protects the UPS from severe impairment during HS. HEK293 cells stably expressing NES-GFPu (a) or NLS-GFPu (b) were either untreated or heat-shocked at 43 °C. Recovery was performed at 37 °C. MG132 (25 µM for 5 h) was used as positive control for UPS inhibition. Cell extracts were blotted against the indicated proteins. c, d HEK293 cells stably expressing NES-GFPu (c) or NLS-GFPu (d) were transfected with nontarget (cntr) or NEDD8 (ND8) siRNAs, or treated with MLN4924 (200 nM). After 48 h cells were heat-shocked for the indicated periods and cell extracts were blotted against the indicated proteins. Bottom right graph: Quantitation of three independent experiments performed as in d ± SD. Two-tailed unpaired Student’s t test, *p < 0.05, **p < 0.01. e Similar experiment as above using the HEK293 NLS-GFPu cells. Twenty-four hours after siRNA transfection, cells were transfected either with pcDNA3-expressing siRNA-resistant NEDD8 or empty vector. f U2OS cells transfected with control or NEDD8 siRNA were either untreated or heat-shocked for 30 min. Cells were harvested and proteasome activity in cytoplasmic or nuclear extracts was measured as described in Methods. Graphs represent the average of the fold change in proteasome activity ± SD from three independent experiments