Figure 3.

pVHL associates with p76 in the cytosol of cells. Lanes 1–4 and 8, VBC complexes were incubated with lysates of [³⁵S]methionine-labeled 786-0 RCC cells and precipitated with M2 resin. The precipitates in lanes 3 and 4 were washed in the presence of 0.5% Sarkosyl or 1 M NaCl, respectively. Lanes 5–7, HeLa cells expressing either rat VHL-F, human VHL-F, or a R167W mutant VHL-F were labeled with [³⁵S]methionine and lysed, and pVHL was immunoprecipitated with M2 antibody. Lanes 9–12, HeLa cells expressing the rat VHL protein were labeled with [³⁵S]methionine and fractionated into a nuclear fraction (600 × g pellet), cytosolic fraction (100,000 × g supernatant), and a membrane fraction (100,000 × g pellet). Total lysate (lane 12) and subcellular fractions were solubilized in Triton X-100 lysis buffer and immunoprecipitated with M2 antibody. All reactions were analyzed on a split SDS/polyacrylamide gel (7% on the top/14% on the bottom) followed by autoradiography.