Figure 5.

NEDD8-modified hCUL-2 is part of the CBC^VHL complex. Human RCC cells stably expressing vector (control), wild-type ^HApVHL, or a naturally occurring mutant ^HApVHL^Δ155 were used for coimmunoprecipitation analysis. pVHL was immunoprecipitated by using anti-HA antibodies, and proteins were analyzed with immunoblots probed with anti-hCUL-2 antibodies. Both hCUL-2-specific bands were excised from a preparative gel blot and subjected to Edman degradation. The obtained sequence of the first 21 cycles derived from the material of the upper band is shown on the Right. The N terminus of hCUL-2 seemed to be blocked, and only the N terminus of the NEDD8 modification was accessible to sequencing. Proteolytic digestion of the hCUL-2 bands, however, yielded peptide sequences corresponding to hCUL-2 (15).