Figure 1.

Type 1, but not type 2 VHL mutants are grossly deficient in HIF-2α regulation. (A) Diagrammatic representation of pVHL, with first and last amino acid residues in bold. Codon 54 represents the start of the pVHL₁₉ product. Mutations used in this study and location of the α and β domains in the reported VHL structure [84] are shown. The location of the elongin binding domain is demarcated with a black box. (B) RCC10 VHL-null cells were stably infected with retroviruses produced with the empty vector with no VHL insert (control) or with wild-type (WT) or mutant pVHL₃₀ expression constructs, as indicated. Cells were grown to confluence and prepared cell lysates were equally loaded and separated by SDS-PAGE. Western blots were performed for VHL, HIF-2α, and GLUT-1. Note that the 3 bands observed for VHL are closely-migrating isoforms of pVHL₃₀ [11], but are not pVHL₁₉, which was not produced by these constructs (data not shown). α-tubulin was also assayed to demonstrate equal loading. *indicates decreased size of VHL deletion protein. (C) A498 VHL-null cells were similarly stably infected and the resulting cell lines were analyzed by western blotting.