Fig. 7.

The HECT E3-ligase HUWE1 is required for stress-induced NEDDylation. a Table showing the SILAC ratio for HUWE1 in the two replicate experiments performed for the aggregate composition upon HS. Heavy, HS; Light, untreated. b H1299 cells were either untreated (DMSO) or exposed to HS (2 h) or to overnight MG132 (5 μM) treatment. Cells were fixed and stained for HUWE1. c U2OS cells were transfected with siRNAs and treated as above. Cell extracts were used for western blotting. d U2OS cells were transfected with siRNAs before plasmid transfection of HA-RPL7 and His₆-NEDD8 as indicated. Cells were exposed to HS (2 h) before Ni-NTA pull-down and western blotting. e, f H1299 cells were siRNA transfected as indicated and either heat-shocked (e) or MG132 (f) treated as before. Staining and quantitation of nuclear aggregates stained with NEDD8 or ubiquitin was performed as before. Scale bars, 10 µm. Values represent the average of three independent experiments ± SD