VHL mutations result in impaired VHL function and increased HIF activation. Mutant proteins are unstable in vitro. (A) Electropherogram showing G/A heterozygosity at base 376 (predicting a change from Aspartic Acid to Asparagine at residue 126) and C/T heterozygosity at base 548 (predicting a change from Serine to Leucine at residue 187). (B) RCC10 cell pools stably expressing either the D126N or S183L mutants show reductions in media pH, reduced levels of glucose and increased levels of lactate (consistent with up-regulated glycolytic metabolism) compared with pools expressing wild type (WT) protein. A nonfunctional VHL mutant (N78S) is presented as a comparison. (C) Western blotting shows increased levels of HIF-1α protein in lysates obtained from D126N and S183L RCC10 cell pools compared with WT RCC10 pools. (D) QPCR analysis of mutant RCC10 pools show increased expression of HIF-1α target genes compared with WT pools. (E) The rate of reduction in protein expression was measured after addition of cycloheximide to inhibit protein translation. This was increased in mutant 786-O cell pools. Quantification of VHL protein (after normalization to HSP90 loading control) is represented graphically in panel F.