PMC

Fig. 1. Conjugation of NEDD8 to Cul2. PC-3 cells were immunoprecipitated (IP) with anti-pVHL antibody. Bound proteins were loaded in a wide well, resolved by electrophoresis and transferred to a membrane. The membrane was cut into strips and immunoblotted (IB) with control, anti-NEDD8 or anti-Cul2 antibodies.

Fig. 6. Impaired neddylation does not globally affect protein polyubiquitylation. ts20 and ts41 cells were grown at permissive (P) or non-permissive (NP) temperature for 15 h with or without proteasome inhibitor MG262 (5 µM). Cells were then lysed, resolved by SDS–PAGE and immunoblotted with anti-ubiquitin antibody.

Fig. 3. In vivo neddylation of Cul2. CHO cells with temperature-sensitive APP-BP1 (ts41) were stably transfected with empty expression plasmid (Mock) or plasmids encoding HA-tagged wild-type (WT) or Cul2 K689R. Clones were grown at permissive (P) or non-permissive (NP) temperature for 15 h and immunoprecipitated with anti-HA antibody. Bound proteins were immunoblotted with anti-Cul2 antibody.

Fig. 4. Neddylation linked to VEC-dependent ubiquitylation in mammalian cells. (A) CHO cells with wild-type (v79) or temperature-sensitive APP-BP1 (ts41) or mouse fibroblasts with temperature-sensitive UBE1 (ts20) were grown at permissive (P) or non-permissive (NP) temperature for 15 h and immunoblotted with anti-HIF1α antibody. (B) ts41 CHO cells were grown at permissive or non-permissive temperature in 1 or 21% O₂ for 15 h and immunoblotted with anti-HIF1α antibody. Lane 1 contained recombinant HIF1α. The asterisk indicates unidentified protein. (C) ts41 cells grown at permissive or non-permissive temperature for 15 h were treated with cyclohexamide (CHX; 10 µg/ml) for the indicated times and immunoblotted with anti-HIF1α antibody. (D) ts41 cells grown at permissive or non-permissive temperature for 15 h were immunoblotted with anti-GLUT1 antibody (upper panel) and anti-α-tubulin antibody (lower panel).

Fig. 5. Neddylation linked to SCF-dependent ubiquitylation in mammalian cells. (A) v79 and ts41 CHO cells were exposed to rat TNFα for the indicated time at the permissive (P) or non-permissive (NP) temperature and immunoblotted with anti-IκBα antibody. Note that IκBα migrates as a doublet due to phosphorylation. The asterisk indicates unidentified protein. (B) ts41 CHO cells were grown in the presence of 0.1 or 10% serum for the indicated time at the permissive or non-permissive temperature and immunoblotted with anti-p27 (upper panel) and anti-α-tubulin antibody (lower panel). Following 48 h of serum starvation, 92% of the cells were in G₀/G₁, as determined by fluorescence-activated cell sorter analysis of propidium iodide stained cells (data not shown).

Fig. 2. Dominant-negative hUbc12OH blocks Cul2-dependent ubiquitylation of HIF in vitro. (A) ³⁵S-labeled Cul2 in vitro translate was incubated with a HeLa FII extract and, where indicated, recombinant NEDD8 and a recombinant hUbc. Modified and unmodified Cul2 were immunoprecipitated with an anti-Cul2 antibody, resolved by SDS–PAGE and detected by fluorography. The background level of Cul2/NEDD8 observed in lane 1 likely reflects the presence of rabbit NEDD8 and Ubc12 in the reticulocyte lysate used for in vitro translation. (B) ³⁵S-labeled Gal4-HIF in vitro translate containing the HIF oxygen-dependent degradation domain (ODD) was incubated with S100 extracts that did (WT) or did not (RC) contain pVHL under conditions permissive for in vitro ubiquitylation. Where indicated, recombinant wild-type hUbc12 or dominant-negative hUbc12OH was added (1, 2 or 4 µM as indicated by triangles). Modified and unmodified Gal4-HIF were immunoprecipitated with anti-Gal4 antibody, resolved by SDS–PAGE and detected by fluorography. (C) hUbc12 or hUbc12OH was added to a WT S100 extract as in (B) (4 µM). At the indicated times the status of Cul2 and HA-pVHL in anti-HA immunoprecipitates (IP) was determined by immunoblot analysis (IB).