VHL regulates expression of Neuromedin U. A) RCC10 retroviral cell pools infected with empty vector or expressing wild-type VHL were prepared as described previously . Cells were cultured for 48 hours and RNA expression analysed using an Affymetrix U133 Plus 2.0 human gene expression array. Microarray analysis identified differentially expressed genes by comparing expression levels in VHL defective cells to expression in VHL expressing cells. The five most highly upregulated and down-regulated genes, including NMU are listed in 1a. B) Protein lysates were prepared from VHL defective RCC10 and RCC4 cell lines and sub-lines stably expressing VHL and samples run on a SDS Page gel. Membranes were probed with HIF-1α (BD, San Diego, CA), HIF-2α (Novus Biologicals, Littleton, CO) and α-tubulin (Sigma, St. Louis, MO). In the absence of VHL both HIF-1α and HIF-2α are clearly detected, however in the presence of VHL levels of both HIF-1α and HIF-2α are suppressed. Equal levels of α-tubulin confirm equivalent loading. C) Real-time RT PCR analysis for NMU and the HIF target gene PHD3 was performed using SYBR-Green PCR Master Mix (Abgene, Epsom, United Kingdom). Data presented here has been normalised to expression of β-actin. Cell lines expressing VHL show marked suppression of NMU and PHD3 compared to VHL defective cell lines (*p < 0.01, using student's T test). (Primers: NMU For: 5'-CCTCAAGGATTACAGCCTG-3', NMU Rev: 5'-GTTCCTGAGGCTTTGGTAG-3; PHD3 For F 5'-GATGCTGAAGAAAGGGC-3', PHD3 Rev R 5'- CTGGCAAAGAGAGTATCTG-3'; β-actin For 5'-CCCAGAGCAAGAGAGAGG-3', β-actin Rev 5'-GTCCAGACGCAGGATG-3').