FIG 2.

Destabilization of B-Myb by pVHL. (A) Regulation of B-Myb by cell density and pVHL. 786-O cells (1 × 10⁶) were cultured for 1 day in culture dishes of different diameters as indicated. The resulting cell lysates were subjected to Western blotting with antibodies against B-Myb, HIF-2α, pVHL, or Hsp90. Hsp90 was used as a loading control. (B) The experiment described in panel A was performed using RCC4 cells. (C) Upregulation of B-Myb mRNA in 786-O cells and RCC4 cells cultured sparsely. Total RNA was purified and analyzed by quantitative RT-PCR. (D) Accumulation of B-Myb upon exposure to the proteasome inhibitor MG132. 786-O cells stably expressing wild-type 3×FLAG-pVHL or mutant 3×FLAG-pVHL(1–155) lacking the VHL box or control cells were cultured under confluent conditions for 1 day, followed by exposure to MG132 (10 μM) for 6 h and Western blotting with antibodies against B-Myb, HIF-2α, or pVHL. Ponceau S staining was used as the loading control. (E) 786-O cells were cultured under confluent conditions for 1 day, exposed to MG132 (10 μM) for 6 h, immunoprecipitated (IP) with anti-FLAG antibody, and immunoblotted with anti-B-Myb or anti-FLAG antibody. The asterisk represents a nonspecific band.