FIG. 3.

Strategy to study pVHL-mediated regulation of mRNA translation in RCC cells. (A) Cytoplasmic extracts from 786-0 cells that either lacked (VHL⁻) or expressed (VHL⁺) pVHL were centrifuged through sucrose gradients to obtain fractions of increasing molecular weight. Absorbance at 254 nm served to monitor the presence of ribosomal subunits, ribosomes, and polysomes. Pooled fractions 1 and 2 contained mRNAs that were completely devoid of ribosomes and ribosome components (they constitute the unbound mRNA component); the mRNA in fractions 3 and 4, which typically included the free small and large ribosomal subunits, respectively, were discarded; pooled fractions 5 to 11 contained mRNAs bound to one or several ribosomes (the polysome-bound mRNA component). From each 786-0 cell population, two sets of radiolabeled cDNA were prepared through reverse transcription in the presence of [α-³³P]dCTP: one from unbound mRNA and one from polysome-bound mRNA. Probes were used for hybridization of cDNA arrays (described in reference 5). (B) Representative cDNA arrays to illustrate the hybridization signals corresponding to either unbound mRNA (left) or polysome-bound mRNA (right). Cell fractions and cDNA array analyses were performed three times, each as a completely independent experiment.