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. 1998 Jul 21;95(15):8817–8822. doi: 10.1073/pnas.95.15.8817

Figure 5.

Figure 5

Down-regulation of VEGF and GLUT1 by VHL gene products. 786-0 cells, either untransfected or stably transfected with pCR3, VHLp24(MPR), VHLp18(MEA), or VHL Δ114–178 were assayed for VEGF RNA (A) and GLUT1 protein (B). (A) Northern blot for VEGF. Total RNA was isolated from cell lines (indicated above blot) grown to low confluence (50–70%), and 40 μg of total RNA was loaded per well. Two independent clones of VHLp24(MPR), VHLp18(MEA), and VHL Δ114–178 were assayed. After probing with VEGF, the blot was stripped and rehybridized with a glyceraldehyde-3-phosphate dehydrogenase (GAPD) probe. Probes are indicated to the left of the blot. (B) Anti-GLUT1 Western blot. Each lane was loaded with 25 μg of protein extract from cell lines (indicated above blot). Western blotting was performed with GLUT1 antisera (Alpha Diagnostic International, San Antonio, TX). Two independent clones of pCR3, VHLp24(MPR), and VHLp18(MEA) were assayed. (C) Elongin binding assay. Beads containing 5 μg of GST or GST-VHL fusion proteins (indicated above lanes) were incubated with 20 μl of in vitro translated elongins B and C, washed, separated on an SDS/8–16% polyacrylamide gel, and visualized by fluorography. Positions of the elongin B and C products are indicated to the left.