FIG. 1.

pVHL fractionates with LMW polysomes. (A) Cytoplasmic extracts from RCC 786-0 cells that either lacked (VHL⁻) or expressed (VHL⁺) pVHL were centrifuged through continuous sucrose gradients to obtain fractions of increasing molecular weight. Absorbance at 254 nm served to monitor the presence of ribosomal subunits, ribosomes, and polysomes. Fractions 1 and 2 were completely devoid of ribosomes and ribosome components, fractions 3 and 4 typically included the free small and large ribosomal subunits, respectively, and fractions 5 to 11 contained polysomes of increasing weight. (B) Representative Western blot analysis to monitor pVHL abundance in each of the continuous fractions prepared from sucrose gradients in both pVHL-expressing and pVHL-deficient cells; equal volumes were loaded from each fraction. To monitor the quality of the protein preparations, blots were stripped and rehybridized using an antibody that recognizes β-actin.